Process and system for obtaining botulinum neurotoxin

ABSTRACT

Rapid, animal protein free, chromatographic processes and systems for obtaining high potency, high yield botulinum neurotoxin for research, therapeutic and cosmetic use.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 13/344,399,filed Jan. 5, 2012, which is a continuation of application Ser. No.12/502,181, filed Jul. 13, 2009. The entire contents of each are hereinincorporated by reference in their entireties.

BACKGROUND

The present invention relates to systems and processes for obtaining aClostridial neurotoxin, methods for making a pharmaceutical compositiontherefrom and to therapeutic and cosmetic uses of the pharmaceuticalcomposition so made. In particular, the present invention relates to arapid, animal protein free, chromatographic process and system forobtaining a high potency, high purity, and high yield biologicallyactive botulinum neurotoxin.

A pharmaceutical composition suitable for administration to a human oranimal for a therapeutic, diagnostic, research or cosmetic purposecomprises an active ingredient and one or more excipients, buffers,carriers, stabilizers, tonicity adjusters, preservatives and/or bulkingagents. The active ingredient in a pharmaceutical composition can be abiologic such as a botulinum neurotoxin. Known methods (such as theSchantz method) for obtaining a botulinum neurotoxin useful as theactive ingredient in a pharmaceutical composition are multi-weekculturing, fermentation and purification processes which useanimal-derived proteins, such as meat broth and casein used in cultureand fermentation media, and animal derived purification enzymes.Administration to a patient of a pharmaceutical composition made throughuse of animal derived products can entail risk of administeringpathogens or an infectious agent, such as a prion. Additionally, knownanimal protein free methods for obtaining a botulinum toxin are alsotime-consuming processes (i.e. take more than a week to complete) withnumerous upstream (culturing and fermentation) and downstream(purification) steps, and yet still result in obtaining a botulinumneurotoxin with detectable impurities.

Botulinum Toxin

The genus Clostridium has more than one hundred and twenty sevenspecies, grouped by morphology and function. The anaerobic, grampositive bacterium Clostridium botulinum produces a potent polypeptideneurotoxin, botulinum toxin (synonymously “toxin”), which causes aneuroparalytic illness in humans and animals known as botulism. Symptomsof botulinum toxin intoxication can progress from difficulty walking,swallowing, and speaking to paralysis of the respiratory muscles anddeath.

One unit of botulinum toxin is defined as the LD₅₀ upon intraperitonealinjection into female Swiss Webster mice weighing about 18-20 gramseach. One unit of botulinum toxin is the amount of botulinum toxin thatkills 50% of a group of female Swiss Webster mice. Seven generallyimmunologically distinct botulinum neurotoxins have been characterized,these being respectively botulinum neurotoxin serotypes A, B, C₁, D, E,F and G each of which is distinguished by neutralization withtype-specific antibodies. The different serotypes of botulinum toxinvary in the animal species that they affect and in the severity andduration of the paralysis they evoke. The botulinum toxins apparentlybind with high affinity to cholinergic motor neurons and translocateinto the neuron and block the presynaptic release of acetylcholine.

Botulinum toxins have been used in clinical settings for the treatmentof e.g. neuromuscular disorders characterized by hyperactive skeletalmuscles. Botulinum toxin type A has been approved by the U.S. Food andDrug Administration (FDA) for the treatment of essential blepharospasm,strabismus and hemifacial spasm in patients over the age of twelve,cervical dystonia, glabellar line (facial) wrinkles and for treatinghyperhydrosis. The FDA has also approved a botulinum toxin type B forthe treatment of cervical dystonia.

Although all the botulinum toxins serotypes apparently inhibit releaseof the neurotransmitter acetylcholine at the neuromuscular junction,they do so by affecting different neurosecretory proteins and/orcleaving these proteins at different sites. Botulinum toxin type A is azinc endopeptidase which can specifically hydrolyze a peptide linkage ofthe intracellular, vesicle-associated protein (VAMP, also calledsynaptobrevin) 25 kiloDalton (kDa) synaptosomal associated protein(SNAP-25). Botulinum type E also cleaves SNAP-25 but targets differentamino acid sequences within this protein, as compared to botulinum toxintype A. Botulinum toxin types B, D, F and G act on VAMP with eachserotype cleaving the protein at a different site. Finally, botulinumtoxin type C₁ has been shown to cleave both syntaxin and SNAP-25. Thesedifferences in mechanism of action may affect the relative potencyand/or duration of action of the various botulinum toxin serotypes.

The molecular weight of the active botulinum toxin protein molecule(also known as “pure toxin” or as the “neurotoxic component”) from abotulinum toxin complex, for all seven of the known botulinum toxinserotypes, is about 150 kDa. Interestingly, the botulinum toxins arereleased by Clostridial bacterium as complexes comprising the 150 kDaneurotoxic component along with one or more associated non-toxinproteins. Thus, the botulinum toxin type A complex can be produced byClostridial bacterium as 900 kDa, 500 kDa and 300 kDa forms (approximatemolecular weights). Botulinum toxin types B and C₁ are apparentlyproduced as only a 500 kDa complex. Botulinum toxin type D is producedas both 300 kDa and 500 kDa complexes. Finally, botulinum toxin types Eand F are produced as only approximately 300 kDa complexes. Thecomplexes (i.e. molecular weight greater than about 150 kDa) containhemagglutinin (HA) proteins and a non-toxin non-hemagglutinin (NTNH)protein. Thus, a botulinum toxin complex can comprise a botulinum toxinmolecule (the neurotoxic component) and one or more HA proteins and/orNTNH protein. These two types of non-toxin proteins (which along withthe botulinum toxin molecule can comprise the relevant neurotoxincomplex) may act to provide stability against denaturation to thebotulinum toxin molecule and protection against digestive acids whentoxin is ingested. Additionally, it is possible that the larger (greaterthan about 150 kDa molecular weight) botulinum toxin complexes mayresult in a slower rate of diffusion of the botulinum toxin away from asite of intramuscular injection of a botulinum toxin complex. Thesuccess of botulinum toxin type A to treat a variety of clinicalconditions has led to interest in other botulinum toxin serotypes. Thus,at least botulinum toxins types, A, B, E and F have been used clinicallyin humans. Additionally, a formulation of the neurotoxic component (i.e.without the associated non-toxin proteins) is sold in Europe under thetradename XEOMIN (Merz Pharmaceuticals, Frankfurt, Germany).

The botulinum toxin type A is known to be soluble in dilute aqueoussolutions at pH 4-6.8. At pH above about 7 the stabilizing non-toxinproteins dissociate from the neurotoxin, resulting in a gradual loss oftoxicity, particularly as the pH and temperature rise (Schantz E. J., etal Preparation and characterization of botulinum toxin type A for humantreatment (in particular pages 44-45), being chapter 3 of Jankovic, J.,et al, Therapy with Botulinum Toxin, Marcel Dekker, Inc, 1994).

As with enzymes generally, the biological activities of the botulinumtoxins (which are intracellular peptidases) are dependant, at least inpart, upon their three dimensional conformation. Dilution of the toxinfrom milligram quantities to a solution containing nanograms permilliliter presents significant difficulties, such as, for example,tendency for toxin to adhere to surfaces and thus reduce the amount ofavailable toxin. Since the toxin may be used months or years after thetoxin containing pharmaceutical composition is formulated, the toxin isstabilized with a stabilizing agent such as albumin, sucrose, trehaloseand/or gelatin.

A commercially available botulinum toxin containing pharmaceuticalcomposition is sold under the trademark BOTOX® (botulinum toxin type Apurified neurotoxin complex) available commercially from Allergan, Inc.,of Irvine, Calif. Each 100 unit vial of BOTOX® consists of about 5 ng ofpurified botulinum toxin type A complex, 0.5 mg human serum albumin, and0.9 mg sodium chloride, vacuum-dried form and intended forreconstitution with sterile normal saline without a preservative (0.9%sodium chloride injection). Other commercially available, botulinumtoxin-containing pharmaceutical compositions include Dysport®(Clostridium botulinum type A toxin hemagglutinin complex with humanserum albumin and lactose in the botulinum toxin pharmaceuticalcomposition), available from Ipsen Limited, Berkshire, U.K. as a powderto be reconstituted with 0.9% sodium chloride before use), and MyoBloc™(an injectable solution comprising botulinum toxin type B, human serumalbumin, sodium succinate, and sodium chloride at about pH 5.6,available from Solstice Neurosciences of San Diego, Calif. Theneurotoxic component (the 150 kDa toxin molecule) and botulinum toxincomplexes (300 kDa to 900 kDa) can be obtained from, for example, ListBiological Laboratories, Inc., Campbell, Calif.; the Centre for AppliedMicrobiology and Research, Porton Down, U.K.; Wako (Osaka, Japan), aswell as from Sigma Chemicals of St Louis, Mo.

Animal protein free and/or chromatographic methods for obtaining abotulinum neurotoxin are disclosed in U.S. Pat. Nos. 7,445,914;7,452,697; 7,354,740; 7,160,699; 7,148,041, and; 7,189,541. Also ofinterest are U.S. patent application Ser. Nos. 11/609,449 entitled“Media for Clostridium Bacterium”, filed Dec. 12, 2006; 12/098,896entitled “Animal Product Free Media and Processes for Obtaining aBotulinum Toxin”, filed Apr. 7, 2008; 11/932,689 entitled“Chromatographic Method and System for Purifying a Botulinum Toxin”,filed Oct. 31, 2007; 11/932,789 entitled “Chromatographic Method andSystem for Purifying a Botulinum Toxin” filed Oct. 31, 2007, and;12/234,537, entitled “Animal Product Free Media And Processes ForObtaining A Botulinum Toxin”, filed Sep. 19, 2008.

Botulinum toxin for use in a pharmaceutical composition can be obtainedby anaerobic fermentation of Clostridium botulinum using the well knownSchantz process (see e.g. Schantz E. J., et al., Properties and use ofbotulinum toxin and other microbial neurotoxins in medicine, MicrobiolRev 1992 March; 56(1):80-99; Schantz E. J., et al., Preparation andcharacterization of botulinum toxin type A for human treatment, chapter3 in Jankovic J, ed. Neurological Disease and Therapy. Therapy withbotulinum toxin (1994), New York, Marcel Dekker; 1994, pages 41-49, and;Schantz E. J., et al., Use of crystalline type A botulinum toxin inmedical research, in: Lewis GE Jr, ed. Biomedical Aspects of Botulism(1981) New York, Academic Press, pages 143-50). The Schantz process forobtaining a botulinum toxin makes use of animal products for example asreagents and as part of the culture and fermentation media.

A number of steps are required to make a Clostridial toxinpharmaceutical composition suitable for administration to a human oranimal for a therapeutic, diagnostic, research or cosmetic purpose.These steps can include obtaining a purified Clostridial toxin and thencompounding the purified Clostridial toxin. A first step can be to plateand grow colonies of Clostridial bacteria, typically on blood agarplates, in an environment conducive to anaerobic bacterial growth, suchas in a warm anaerobic atmosphere. This step allows Clostridial colonieswith desirable morphology and other characteristics to be obtained. In asecond step selected Clostridial colonies can be fermented in a firstsuitable medium and if additionally desired, into a second fermentationmedium. After a certain period of fermentation, the Clostridial bacteriatypically lyse and release Clostridial toxin into the medium. Thirdly,the medium can be purified so as to obtain a bulk toxin. Typicallymedium purification to obtain bulk toxin is carried out using, amongother reagents, animal-derived enzymes, such as DNase and RNase, whichare used to degrade and facilitate removal of nucleic acids. Theresulting bulk toxin can be a highly purified toxin with a particularspecific activity. After stabilization in a suitable buffer, the bulktoxin can be compounded with one or more excipients to make aClostridial toxin pharmaceutical composition suitable for administrationto a human. The Clostridial toxin pharmaceutical composition cancomprise a Clostridial toxin as an active pharmaceutical ingredient(API). The pharmaceutical composition can also include one or moreexcipients, buffers, carriers, stabilizers, preservatives and/or bulkingagents.

The Clostridium toxin fermentation step can result in a fermentationmedium solution that contains whole Clostridium bacteria, lysedbacteria, culture medium nutrients and fermentation by-products.Filtration of this culture solution so as to remove gross elements, suchas whole and lysed bacteria, provides a harvest/clarified medium. Theclarified medium comprises a Clostridial toxin and various impuritiesand is processed to obtain a concentrated Clostridial toxin, which iscalled bulk toxin.

Fermentation and purification processes for obtaining a bulk Clostridialtoxin using one or more animal derived products (such as the milk digestcasein, DNase and RNase) are known. An example of such a knownnon-animal product free (“NAPF”) process for obtaining a botulinum toxincomplex is the Schantz process and modifications thereto. The Schantzprocess (from initial plating, cell culture through to fermentation andtoxin purification) makes use of a number of products derived fromanimal sources such as, for example, animal derived Bacto Cooked Meatmedium in the culture vial, Columbia Blood Agar plates for colony growthand selection, and casein in the fermentation media. Additionally, theSchantz bulk toxin purification process makes use of DNase and RNasefrom bovine sources to hydrolyze nucleic acids present in the toxincontaining fermentation medium. Concerns have been expressed regarding apotential for a viral and transmissible spongiform encephalopathy (TSE),such as a bovine spongiform encephalopathy (BSE), contamination whenanimal products are used in a process for obtaining an API and/or in aprocess for making (compounding) a pharmaceutical composition using suchan API.

A fermentation process for obtaining a tetanus toxoid that uses reducedamounts of animal-derived products (referred to as animal product freeor “APF” fermentation processes; APF encompasses animal protein free) isknown, see e.g. U.S. Pat. No. 6,558,926. An APF fermentation process forobtaining a Clostridial toxin, has the potential advantage of reducingthe (the already very low) possibility of contamination of the ensuingbulk toxin with viruses, prions or other undesirable elements which canthen accompany the active pharmaceutical ingredient, Clostridial toxin,as it is compounded into a pharmaceutical composition for administrationto humans.

Chromatography, such as column chromatography for example, can be usedto separate a particular protein (such as a botulinum neurotoxin) from amixture of proteins, nucleic acids, cell debris, etc. in a process knownas fractionation or purification. The protein mixture typically passesthrough a glass or plastic column containing, for example, a solid,often porous media (often referred to as beads or resin). Differentproteins and other compounds pass through the matrix at different ratesbased on their specific chemical characteristics and the way in whichthese characteristics cause them to interact with the particularchromatographic media utilized.

The choice of media determines the type of chemical characteristic bywhich the fractionation of the proteins is based. There are four basictypes of column chromatography; ion-exchange, gel filtration, affinityand hydrophobic interaction. Ion-exchange chromatography accomplishesfractionation based on surface electrostatic charge using a columnpacked with small beads carrying either a positive or a negative charge.In gel filtration chromatography, proteins are fractionated based ontheir size. In affinity chromatography, proteins are separated based ontheir ability to bind to specific chemical groups (ligand) attached tobeads in the column matrix. Ligands can be biologically specific for atarget protein. Hydrophobic interaction chromatography accomplishesfractionation based on surface hydrophobicity.

Column chromatography to purify (fractionate) a Clostridial toxin iswell known. See for example the following publications:

1. Ozutsumi K., et al, Rapid, simplified method for production andpurification of tetanus toxin, App & Environ Micro, April 1985, p939-943, vol 49, no. 4. (1985) discloses use of high pressure liquidchromatography (HPLC) gel filtration to purify tetanus toxin.2. Schmidt J. J., et al., Purification of type E botulinum neurotoxin byhigh-performance ion exchange chromatography, Anal Biochem 1986 July;156(1):213-219 discloses use of size exclusion chromatography or ionexchange chromatograph to purify botulinum toxin type E. Also disclosedis use of protamine sulfate instead of ribonuclease (RNase).3. Simpson L. L., et al., Isolation and characterization of thebotulinum neurotoxins Simpson L L; Schmidt J J; Middlebrook J L, In:Harsman S, ed. Methods in Enzymology. Vol. 165, Microbial Toxins: Toolsin Enzymology San Diego, Calif.: Academic Press; vol 165: pages 76-85(1988) discloses purification of botulinum neurotoxins using gravityflow chromatography, HPLC, capture steps using an affinity resin, sizeexclusion chromatography, and ion (anion and cation) exchangechromatography, including use of two different ion exchange columns.Various Sephadex, Sephacel, Trisacryl, S and Q columns are disclosed.4. Zhou L., et al., Expression and purification of the light chain ofbotulinum neurotoxin A: A single mutation abolishes its cleavage ofSNAP-25 and neurotoxicity after reconstitution with the heavy chain,Biochemistry 1995; 34(46):15175-81 (1995) discloses use of an amyloseaffinity column to purify botulinum neurotoxin light chain fusionproteins.5. Kannan K., et al., Methods development for the biochemical assessmentof Neurobloc (botulinum toxin type B), Mov Disord 2000; 15(Suppl 2):20(2000) discloses use of size exclusion chromatography to assay abotulinum toxin type B.6. Wang Y-c, The preparation and quality of botulinum toxin type A forinjection (BTXA) and its clinical use, Dermatol Las Faci Cosm Surg 2002;58 (2002) discloses ion exchange chromatography to purify a botulinumtoxin type A. This reference discloses a combination of precipitationand chromatography techniques.7. Johnson S. K., et al., Scale-up of the fermentation and purificationof the recombination heavy chain fragment C of botulinum neurotoxinserotype F, expressed in Pichia pastoris, Protein Expr and Purif 2003;32:1-9 (2003) discloses use of ion exchange and hydrophobic interactioncolumns to purify a recombinant heavy chain fragment of a botulinumtoxin type F.8. Published U.S. patent application 2003 0008367 A1 (Oguma) disclosesuse of ion exchange and lactose columns to purify a botulinum toxin.

The purification methods summarized above relate to small-scalepurification of the neurotoxic component of a botulinum toxin complex(i.e. the approximately 150 kDa neurotoxic molecule), or a specificcomponent of the neurotoxic component, as opposed to purification of theentire 900 kDa botulinum toxin complex.

Furthermore, existing processes, including commercial scale processes,for obtaining a botulinum toxin suitable for compounding into abotulinum toxin pharmaceutical composition typically include a series ofprecipitation steps to separate the toxin complex from impurities thataccompany the botulinum toxin from the fermentation process. Notably,precipitation techniques are widely used in the biopharmaceuticalindustry to purification a botulinum toxin. For example, cold alcoholfractionation (Cohn's method) or precipitation is used to remove plasmaproteins. Unfortunately, previous precipitation techniques for purifyinga botulinum toxin have the drawbacks of low resolution, lowproductivity, difficulty of operation, difficulty to control and/orvalidate and/or difficulty to scale-up or scale-down. Previouslypublished U.S. patent application Ser. No. 11/452,570, published Oct.12, 2006, discloses steps such as centrifugation, acid precipitation,ethanol precipitation, acidification steps, and ammonium sulfateprecipitation utilized in various animal-protein free and NAPF processes(for a detailed discussion, see U.S. Published Patent App. No.2006/0228780, herein incorporated by reference in its entirety). Somedistinctions between a non-animal protein free process and an animalprotein free processes for obtaining a botulinum neurotoxin are showntherein.

What are needed therefore are rapid, relatively smaller scale yet highyield systems and processes for obtaining high purity, highly potentbotulinum neurotoxin, which can be used for research purposes and/or tomake a pharmaceutical composition.

SUMMARY

The present invention meets this need and provides high purity, highlypotent botulinum neurotoxins obtainable by rapid, smaller scaled,commercially useful, high yield, animal protein free, chromatographicsystems and processes. The resultant botulinum neurotoxin is useful formaking a pharmaceutical composition. The Clostridial toxin obtained bythe practice of our invention is preferably a botulinum neurotoxin andmost preferably a botulinum neurotoxin type A complex of about 900 kDaor the 150 kDa neurotoxic component therefrom. Our invention does notrequire NAPF reagents, such as DNase and RNase.

DEFINITIONS

The following words and terms used herein have the followingdefinitions.

“About” means that the item, parameter or term so qualified encompassesa range of plus or minus ten percent above and below the value of thestated item, parameter or term.

“Administration,” or “to administer” means the step of giving (i.e.administering) a pharmaceutical composition or active ingredient to asubject. The pharmaceutical compositions disclosed herein are “locallyadministered” by e.g. intramuscular (i.m.), intradermal, subcutaneousadministration, intrathecal administration, intracranial,intraperitoneal (i.p.) administration, topical (transdermal) andimplantation (i.e. of a slow-release device such as polymeric implant orminiosmotic pump) routes of administration.

“Animal product free” or “substantially animal product free”encompasses, respectively, “animal protein free” or “substantiallyanimal protein free” and means the absence or substantial absence ofblood derived, blood pooled and other animal derived products orcompounds. “Animal” means a mammal (such as a human), bird, reptile,fish, insect, spider or other animal species. “Animal” excludesmicroorganisms, such as bacteria. Thus, an APF medium or process or asubstantially APF medium or process within the scope of the presentinvention can include a botulinum toxin or a Clostridial botulinumbacterium. For example, an APF process or a substantially APF processmeans a process which is either substantially free or essentially freeor entirely free of animal-derived proteins, such as immunoglobulins,meat digest, meat by products and milk or dairy products or digests.

“Botulinum toxin” or “botulinum neurotoxin: means a neurotoxin producedby Clostridium botulinum, as well as modified, recombinant, hybrid andchimeric botulinum toxins. A recombinant botulinum toxin can have thelight chain and/or the heavy chain thereof made recombinantly by anon-Clostridial species. “Botulinum toxin,” as used herein, encompassesthe botulinum toxin serotypes A, B, C, D, E, F and G. “Botulinum toxin,”as used herein, also encompasses both a botulinum toxin complex (i.e.the 300, 600 and 900 kDa complexes) as well as pure botulinum toxin(i.e. the about 150 kDa neurotoxic molecule), all of which are useful inthe practice of the present invention. “Purified botulinum toxin” meansa pure botulinum toxin or a botulinum toxin complex that is isolated, orsubstantially isolated, from other proteins and impurities which canaccompany the botulinum toxin as it is obtained from a culture orfermentation process. Thus, a purified botulinum toxin can have at least90%, preferably more than 95%, and most preferably more than 99% of thenon-botulinum toxin proteins and impurities removed. The botulinum O₂and C₃ cytotoxins, not being neurotoxins, are excluded from the scope ofthe present invention.

“Clostridial neurotoxin” means a neurotoxin produced from, or native to,a Clostridial bacterium, such as Clostridium botulinum, Clostridiumbutyricum or Clostridium beratti, as well as a Clostridial neurotoxinmade recombinantly by a non-Clostridial species.

“Entirely free” (“consisting of” terminology) means that within thedetection range of the instrument or process being used, the substancecannot be detected or its presence cannot be confirmed.

“Essentially free” (or “consisting essentially of”) means that onlytrace amounts of the substance can be detected.

“Modified botulinum toxin” means a botulinum toxin that has had at leastone of its amino acids deleted, modified, or replaced, as compared to anative botulinum toxin. Additionally, the modified botulinum toxin canbe a recombinantly produced neurotoxin, or a derivative or fragment of arecombinantly made neurotoxin. A modified botulinum toxin retains atleast one biological activity of the native botulinum toxin, such as,the ability to bind to a botulinum toxin receptor, or the ability toinhibit neurotransmitter release from a neuron. One example of amodified botulinum toxin is a botulinum toxin that has a light chainfrom one botulinum toxin serotype (such as serotype A), and a heavychain from a different botulinum toxin serotype (such as serotype B).Another example of a modified botulinum toxin is a botulinum toxincoupled to a neurotransmitter, such as substance P.

“Pharmaceutical composition” means a formulation in which an activeingredient can be a botulinum toxin. The word “formulation” means thatthere is at least one additional ingredient (such as, for example andnot limited to, an albumin [such as a human serum albumin or arecombinant human albumin] and/or sodium chloride) in the pharmaceuticalcomposition in addition to a botulinum neurotoxin active ingredient. Apharmaceutical composition is therefore a formulation which is suitablefor diagnostic, therapeutic or cosmetic administration (e.g. byintramuscular or subcutaneous injection or by insertion of a depot orimplant) to a subject, such as a human patient. The pharmaceuticalcomposition can be: in a lyophilized or vacuum dried condition, asolution formed after reconstitution of the lyophilized or vacuum driedpharmaceutical composition with saline or water, for example, or; as asolution that does not require reconstitution. The active ingredient canbe one of the botulinum toxin serotypes A, B, C₁, D, E, F or G or abotulinum toxin, all of which can be made natively by Clostridialbacteria. As stated, a pharmaceutical composition can be liquid orsolid, for example vacuum-dried. Exemplary methods for formulating abotulinum toxin active ingredient pharmaceutical composition aredisclosed in published U.S. patent publication 20030118598, filed Nov.5, 2002, herein incorporated by reference in its entirety.

“Substantially free” means present at a level of less than one percentby weight of a culture medium, fermentation medium, pharmaceuticalcomposition or other material in which the weight percent of a substance(such as an animal product, animal protein or animal derived product orprotein) is assessed.

“Therapeutic formulation” means a formulation can be used to treat andthereby alleviate a disorder or a disease and/or symptom associatedthereof, such as a disorder or a disease characterized by hyperactivity(e.g. spasticity) of a peripheral muscle or gland, (e.g. sweat gland).

“Therapeutically effective amount” means the level, amount orconcentration of an agent (e.g. such as a botulinum toxin orpharmaceutical composition comprising botulinum toxin) needed to treat adisease, disorder or condition without causing significant negative oradverse side effects.

“Treat”, “treating”, or “treatment” means an alleviation or a reduction(which includes some reduction, a significant reduction a near totalreduction, and a total reduction), resolution or prevention (temporarilyor permanently) of an disease, disorder or condition, such as a buttockdeformity, so as to achieve a desired therapeutic or cosmetic result,such as by healing of injured or damaged tissue, or by altering,changing, enhancing, improving, ameliorating and/or beautifying anexisting or perceived disease, disorder or condition. A treatmenteffect, such as an alleviating effect from administration of a botulinumneurotoxin may not appear clinically for between 1 to 7 days afteradministration of the botulinum neurotoxin to a patient for example andcan have a duration of effect of from about 1 month to about 1 year orany range of time therebetween, for example, depending upon thecondition and particular case being treated.

Percentages are based on weight per volume unless otherwise noted.APF means animal product/protein freeCV means column volumeDF means diafiltrationELISA means enzyme-linked immunosorbent assay.IAPF, as in “IAPF system” or “IAPF process”, means “improved animalprotein free” system or process. An IAPF system or process includes theuse of either two chromatography media or three chromatography media topurify a botulinum toxin or neurotoxin component, as specificallydetailed herein. Chromatography media includes chromatography resins, asknown in the art. Batches of botulinum neurotoxin obtained by use of twochromatography media are herein designated as IAPF.FAPF, as in “FAPF system” or “FAPF process”, means “further improvedanimal protein free” system or process. Accordingly, FAPF is an IAPFprocess, and a FAPF system or process means that three chromatographymedia are used to purify a botulinum toxin or neurotoxin component.Batches of botulinum neurotoxin obtained by use of three chromatographymedia are herein designated as FAPF.NAPF means non-animal protein freeSDS-PAGE means sodium dodecylsulfate polyacrylamide gel electrophoresisSEC-HPLC means size exclusion high performance liquid chromatographyUF means ultrafiltration

In one embodiment of the invention, a substantially APF chromatographicprocess for obtaining a biologically active botulinum neurotoxin isprovided, the process comprising the following steps of (a) providing asubstantially APF fermentation medium; (b) fermenting Clostridiumbotulinum bacteria in the fermentation medium, and; (c) recovering thebiologically active botulinum neurotoxin from the fermentation medium bycontacting the fermentation medium with an anion exchange chromatographymedia followed by contacting an eluent from the anion exchangechromatography medium with a cation exchange chromatography media, tothereby obtain the biologically active botulinum neurotoxin from thesubstantially APF chromatographic process. In particular embodiments,the process can provide a botulinum neurotoxin that comprises less thanone part per million (ppm) residual nucleic acid which is one nanogramor less of residual nucleic acid for each milligram of the botulinumneurotoxin obtained. In still another aspect, the process is carried outin one week or less.

In one example, media having a ratio of 3:1:1 means a botulinum toxinculture/fermentation medium containing 3% HySoy, 1% HyYeast, and 1%glucose. HySoy (Quest product no. 5×59022) is a source of peptides madeby enzymatic hydrolysis of soy. HyYeast (HyYest, Quest product no.5Z10102 or 5Z10313 is a baker's yeast extract. In another example, mediahaving a ratio of 5:1:1 means a botulinum toxin culture/fermentationmedium containing 5% HySoy, 1% HyYeast, and 1% glucose.

Another embodiment provides a substantially APF chromatographic processfor obtaining a biologically active botulinum neurotoxin type A complex,the process comprising the following sequential steps of culturingClostridium botulinum bacteria in a substantially APF culture medium;fermenting Clostridium botulinum bacteria from the culture medium inabout 2 L to about 75 L of a substantially APF fermentation medium, morepreferably in about 2 L to about 50 L of a substantially APFfermentation medium, even more preferably in about 2 L to about 30 L ofa substantially APF fermentation medium (particular embodiments have atleast one of the culture medium and the fermentation medium including avegetable protein and/or a vegetable protein derivative, for example ahydrolyzed vegetable protein), harvesting the fermentation medium byremoving cellular debris present in the fermentation medium usingfiltration or centrifugation; concentrating the harvested fermentationmedium by filtration, such as by ultrafiltration (UF) for example;diluting the concentrated fermentation medium by adding a buffer.Following dilution with the buffer, a first contacting step isundertaken in which the diluted harvested fermentation medium iscontacted with an anion exchange media so that the biologically activebotulinum neurotoxin becomes captured by the anion exchange media;followed by elution of the captured botulinum neurotoxin from the anionexchange media to thereby obtain a first eluent containing the botulinumtoxin; performing a second contacting step in which the first eluent iscontacted with a cation exchange media to remove impurities from thefirst eluent, to thereby obtain a second eluent containing the botulinumtoxin; followed by processing the second eluent by diafiltration (DF);and filtering the processed second eluent, thereby obtainingbiologically active botulinum neurotoxin type A complex using asubstantially APF chromatographic process. The botulinum neurotoxin typeA complex obtained can have a potency of about 2.0×10⁷ units/mg to about6.0×10⁷ units/mg of botulinum neurotoxin type A complex. In particularexamples botulinum neurotoxin type A complex having a potency of betweenabout 2.4×10⁷ units/mg to about 5.9×10⁷ units/mg, for example, can beobtained.

In a particular embodiment, the process utilizes fermentation mediumcomprising no more than about 5% w/v of a vegetable-derived proteinproduct, no more than about 2% w/v of a yeast extract and no more thanabout 2% w/v glucose, and wherein the pH level of the fermentationmedium is from about 6.5 to about pH 8.0, more preferably from about pH6.8 to about pH 7.6, at the commencement of the fermenting step. In aparticular embodiment, the culturing step is carried out until theoptical density of the culture medium at about 540 nanometers (nm) isbetween about 0.8 absorbance units (AU) and about 4.5 AU. The culturingstep is preferably initiated by introducing a Clostridium botulinum APFworking cell bank content to the culture medium, where the working cellbank content comprises at least about 1×10⁴ colony-forming units,preferably from about 1×10⁴ to about 5×10⁷ colony-forming units ofClostridium botulinum per milliliter of the working cell bank, and wherethe Clostridium botulinum bacterium in the working cell bank have asubstantially uniform morphology. In still yet another embodiment, thefermenting step is carried out for about 60 to 80 hours and until anoptical density of the fermentation medium at about 890 nm decreases tobetween about 0.05 AU to about 0.7 AU. In one aspect, the botulinumneurotoxin obtained by a substantially APF chromatographic processcomprises less than 1 ppm of residual nucleic acid and the process iscarried out in one week or less.

In yet another embodiment, an APF process utilizing chromatography forobtaining a biologically active botulinum neurotoxin is provided,comprising the sequential steps of: (a) adding Clostridium botulinumbacteria from an APF working cell bank to an APF culture medium; (b)culturing the Clostridium botulinum bacteria in the culture medium; (c)fermenting the Clostridium botulinum bacteria from step (b) in an APFfermentation medium until Clostridium botulinum cell lysis occurs; (d)harvesting the fermentation culture to provide a harvested fermentationmedium; (e) subjecting the harvested fermentation medium toconcentration by filtration; (f) diluting the filtered fermentationmedium by addition of a buffer to obtain a diluted fermentation medium;(g) a first contacting step in which the diluted fermentation medium iscontacted with a capture chromatography media, wherein the capturechromatography media is an anion exchange media; (h) a second contactingstep wherein an eluent from the first contacting step is contacted witha polishing chromatography media, wherein the polishing chromatographymedia is a cation exchange media, and (i) filtering eluent from thesecond contacting step, thereby obtaining biologically active botulinumneurotoxin by the improved APF process, wherein the botulinum neurotoxinobtained comprises 1 ppm of residual nucleic acid or less than 1 ppm ofresidual nucleic acid and the process is carried out in one week orless.

In one aspect, a substantially animal product free (APF) chromatographicsystem for obtaining a biologically active botulinum neurotoxin isprovided, comprising a substantially APF fermentation medium,Clostridium botulinum bacteria for fermenting in the fermentationmedium, an anion exchange chromatography medium for recoveringbiologically active botulinum neurotoxin from the fermentation medium,and a cation exchange chromatography medium for recovering furtherbiologically active botulinum neurotoxin from an eluent from the anionexchange chromatography medium, thereby obtaining biologically activebotulinum neurotoxin from a substantially APF chromatography process. Inparticular configurations, the system can further comprise a firstapparatus for anaerobically culturing the Clostridium botulinum bacteriain a substantially APF culture medium, and can further be comprised of asecond apparatus for anaerobically fermenting the Clostridium botulinumbacteria in the substantially APF fermentation medium, wherein theClostridium botulinum bacteria are obtained from the first apparatus.For clarification, the system can include a harvesting apparatus forremoving cellular debris from the fermentation medium obtained from thesecond apparatus, to thereby provide a harvested fermentation medium.The harvested fermentation medium can be passed through a concentrationand diluting apparatus to concentrate then subsequently dilute theharvested fermentation medium.

In a particular example, the system can also include hydrophobicinteraction medium for recovering further purified biologically activebotulinum neurotoxin from an eluent from the cation exchangechromatography medium. Additionally, a filtration apparatus for reducingbioburden in the obtained biologically active botulinum neurotoxin canalso make up the system, for reducing the bioburden of the biologicallyactive botulinum neurotoxin obtained by utilizing either two or threechromatography medium. In a specific example, an anaerobic chamberhaving an integrated high efficiency particulate air filter within itsworkspace, for culturing Clostridium botulinum bacteria in thesubstantially APF culture medium, can be utilized. Exemplary systems canprovide botulinum neurotoxin having a potency of at least about 2.0×10⁷units/mg of botulinum neurotoxin and the botulinum neurotoxin obtainedcomprises one ng or less than one ng of residual nucleic acid for eachmg of the botulinum neurotoxin obtained. In particular embodiments, thesubstantially APF fermentation medium is provided in an amount of fromabout 2 L to about 75 L; and from about 200 mL to about 1 L ofsubstantially APF culture medium is utilized.

In another aspect of our invention, a substantially APF system usingchromatography for obtaining a biologically active botulinum neurotoxinis provided, the system comprising a first apparatus for culturingClostridium botulinum bacteria, the first apparatus capable ofcontaining a substantially APF culture medium; a second apparatus forfermenting Clostridium botulinum bacteria which have been cultured inthe first apparatus, the second apparatus capable of containing asubstantially APF fermentation medium; a third apparatus for harvestingthe fermentation medium; a fourth apparatus for concentrating theharvested fermentation medium and diluting the filtered fermentationmedium; a fifth apparatus for carrying out a first purification of thebotulinum neurotoxin from the harvested medium, wherein the fifthapparatus comprises an anion exchange chromatography media, therebyobtaining a first purified botulinum neurotoxin; and a sixth apparatusfor carrying out a second purification of the botulinum neurotoxinwherein the sixth apparatus comprises a cation exchange chromatographymedia, to thereby obtain a second purified botulinum neurotoxin, whereinthe botulinum neurotoxin obtained has a potency of at least about2.0×10⁷ units/mg of botulinum neurotoxin to about 5.9×10⁷ units/mg ofbotulinum neurotoxin, the botulinum neurotoxin obtained comprises one ngor less than one ng of residual nucleic acid for each mg of thebotulinum neurotoxin obtained and the process is carried out in one weekor less. In particular embodiments, the botulinum neurotoxin obtainedcan have a potency of at least 4.4×10⁷ units/mg of botulinum neurotoxin.In a particular embodiment of the system, the system can furthercomprise a seventh apparatus for carrying out a further purification ofthe botulinum neurotoxin obtained from the sixth apparatus, wherein theseventh apparatus comprises a hydrophobic interaction media, therebyobtaining a third purified botulinum neurotoxin. In an additionalembodiment, the system can further comprise an eighth apparatuscomprising a membrane for filtering eluent from the seventh apparatus.

Another aspect of our invention includes a substantially APFchromatographic system for obtaining a biologically active botulinumneurotoxin comprising a first apparatus for anaerobic culturingClostridium botulinum bacteria, the first apparatus capable ofcontaining from about 200 mL to about 1 L of a substantially APF culturemedium; a second apparatus comprising an anaerobic chamber having anintegrated high efficiency particulate air filter within the chambercapable of containing the first apparatus; a third apparatus foranaerobic fermentation of Clostridium botulinum bacteria which has beencultured in the first apparatus, the third apparatus capable ofcontaining from about 2 L to about 75 L of a substantially APFfermentation medium, preferably from about 2 L to about 30 L of asubstantially APF fermentation medium and including at least onedisposable probe selected from the group consisting of areduction-oxidation probe, a pH probe and a turbidity probe; a fourthapparatus for harvesting the fermentation medium; a fifth apparatus forconcentrating the harvested fermentation medium and diluting thefiltered fermentation medium; a sixth apparatus for carrying out a firstpurification of botulinum neurotoxin obtained from the harvestedfermentation medium, the sixth apparatus comprising an anion exchangechromatography media, thereby obtaining a first purified botulinumneurotoxin; a seventh apparatus for carrying out a second purificationof the botulinum neurotoxin the seventh apparatus comprising a cationexchange chromatography media, thereby obtaining a second purifiedbotulinum neurotoxin; an eighth apparatus for carrying out a thirdpurification of the second purified botulinum neurotoxin, the eighthapparatus comprising hydrophobic interaction media to thereby obtain athird purified botulinum neurotoxin; and a ninth apparatus for filteringthe third purified botulinum neurotoxin, the ninth apparatus comprisinga filtration membrane, wherein the botulinum neurotoxin obtained has apotency of about 2.4×10⁷ units/mg of botulinum neurotoxin to about5.9×10⁷ units/mg of botulinum neurotoxin, the botulinum neurotoxinobtained comprises one ng or less than one ng of residual nucleic acidfor each mg of the botulinum neurotoxin obtained and the process iscarried out in one week or less. In accordance with these processes, abiologically active botulinum neurotoxin is thereby obtained, and inparticular examples, the botulinum neurotoxin obtained has a potency ofat least about 4.4×10⁷ units/mg of botulinum neurotoxin.

In accordance with processes and systems herein disclosed, biologicallyactive botulinum neurotoxin is thereby obtained. In particularembodiments, the biologically active botulinum neurotoxin obtained bythe process and systems herein disclosed has a molecular weight of about900 kDa.

Our invention further includes a method for making a substantially APFpharmaceutical composition in which the active ingredient is abiologically active botulinum neurotoxin, the method comprising thesteps of: (a) obtaining a biologically active botulinum neurotoxin by:(i) providing a fermentation medium which is substantially free of ananimal product; (ii) fermenting Clostridium botulinum bacteria in thefermentation medium, and; (iii) recovering the biologically activebotulinum neurotoxin from the fermentation medium, using an anionexchange chromatography media followed by use of a cation exchangechromatography media, wherein the botulinum neurotoxin recovered has apotency of at least about 2.0×10⁷ units/mg of botulinum neurotoxin,preferably about 2.4×10⁷ units/mg of botulinum neurotoxin to about5.9×10⁷ units/mg of botulinum neurotoxin, in some embodiments at leastabout 4.4×10⁷ units/mg of botulinum neurotoxin, the botulinum neurotoxincomprises one ng or less than one ng of residual nucleic acid for eachmg of the botulinum neurotoxin, and steps (i) to (iii) are completed inone week or less, and; (b) compounding the botulinum neurotoxin with atleast one suitable excipient, thereby making a substantially APFpharmaceutical composition. In a particular embodiment, the compoundingstep comprises the step of drying the botulinum neurotoxin by a processselected from the group of processes consisting of freeze drying,lyophilization and vacuum drying and wherein the suitable excipient isselected from the group consisting of albumin, human serum albumin,recombinant human serum albumin, gelatin, sucrose, trehalose,hydroxyethyl starch, collagen, lactose, sucrose sodium chloride,polysaccharide, caprylate, polyvinylpyrrolidone and sodium. Accordingly,one aspect our invention also provides substantially APF pharmaceuticalcompositions made by compounding the biologically active botulinumneurotoxin obtained by the processes and systems herein disclosed.

Additionally, our invention also includes a method for treating acondition in a patient, the method comprising the step of administeringto the patient a therapeutically effective amount of a pharmaceuticalcomposition made by methods for making a substantially APFpharmaceutical composition in which the active ingredient is abiologically active botulinum neurotoxin obtained by the APF processes(i.e. IAPF and FAPF processes) herein disclosed. Examples of conditionsto be treated are selected from the group consisting of a headache, amigraine headache, tension headache, a sinus headache, a cervicogenicheadache, a sweating disorder, axillary hyperhidrosis, palmarhyperhidrosis, plantar hyperhidrosis, Frey's syndrome, a hyperkineticskin line, a facial wrinkle, glabellar lines, crow's feet, marionettelines, a nasolabial fold, a skin disorder, achalasia, strabismus,chronic anal fissure, blepharospasm, musculoskeletal pain, fibromyalgia,pancreatitis, tachycardia, prostatic enlargement, prostatitis, urinaryretention, urinary incontinence, overactive bladder, hemifacial spasm,tremors, myoclonus, gastrointestinal disorders, diabetes, sialorrhea,detrusor-sphincter dyssynergia, post stroke spasticity, wound healing,juvenile cerebral palsy, smooth muscle spasm, restenosis, a focaldystonia, epilepsy, cervical dystonia, thyroid disorder, hypercalcemia,an obsessive compulsive disorder, arthritic pain, Raynaud's syndrome,striae distensae, peritoneal adhesion, vasospasms, rhinorrhea, musclecontracture, an injured muscle, laryngeal dystonia, writer's cramp andcarpel tunnel syndrome, for example.

In one embodiment, a method for treating a condition in a patient, themethod comprising the step of locally administering to the patient aneffective amount of a substantially APF pharmaceutical composition madeby a method including the steps of:

(a) obtaining a biologically active botulinum neurotoxin by (i)providing a fermentation medium which is substantially free of an animalproduct; (ii) fermenting Clostridium botulinum bacteria in thefermentation medium, and; (iii) recovering the biologically activebotulinum neurotoxin from the fermentation medium, using an anionexchange chromatography media followed by use of a cation exchangechromatography media, wherein the botulinum neurotoxin recovered has apotency of at least about 2.0×10⁷ units/mg of botulinum neurotoxin, thebotulinum neurotoxin comprises one ng or less than one ng of residualnucleic acid for each mg of the botulinum neurotoxin, and steps (i) to(iii) are completed in one week or less, and;(b) compounding the botulinum neurotoxin with at least one suitableexcipient, thereby making a substantially APF pharmaceuticalcomposition, whereby local administration of the substantially APFpharmaceutical composition treats the condition.

Local administration of therapeutically effective amounts of apharmaceutical compositions, comprising a biologically active botulinumneurotoxin provided by the IAPF process/systems/method herein disclosed,can be repeated at intervals of from about 2 months to about 6 months orat intervals of about 2 months to about 3 months, for example. Exemplaryuseful dosages locally administered to the patient of a therapeuticallyeffective amount of a substantially APF pharmaceutical composition madein accordance with the present disclosure, can have botulinum neurotoxinunit amounts of between about 0.01 unit and about 10,000 units. Inparticular instances, the botulinum neurotoxin is administered in anamount of between about 0.01 unit and about 3000 units. In particularexamples, the biologically active botulinum neurotoxin that is theactive pharmaceutical ingredient in the pharmaceutical composition isbotulinum neurotoxin type A or type B, for example.

Our invention includes a substantially APF process, utilizingchromatography, for obtaining a biologically active botulinumneurotoxin. The process can comprise the sequential steps of providing asubstantially APF fermentation medium, followed by fermentingClostridium botulinum bacteria in the fermentation medium and recoveringthe biologically active botulinum neurotoxin from the fermentationmedium using an anion exchange chromatography media followed by use of acation exchange chromatography media to thereby obtain the biologicallyactive botulinum neurotoxin from the substantially APF chromatographicprocess. The recovering step can also include the use of a hydrophobicinteraction media after the use of cation exchange chromatography media.The biologically active botulinum neurotoxin obtained can be a botulinumneurotoxin complex or a botulinum toxin neurotoxic component isolatedtherefrom with a molecular weight of about 150 kDa free of thecomplexing proteins of a botulinum toxin complex. The APF processes(utilizing 2-columns (IAPF), e.g. anion followed by cationchromatography; or 3-columns (FAPF), e.g. anion followed by cationfollowed by hydrophobic interaction chromatography) can be used toobtain a biologically active botulinum neurotoxin such as botulinumneurotoxins type A, B, C₁, D, E, F and G. The botulinum neurotoxinobtained is preferably a botulinum neurotoxin type A complex.

In one aspect of our invention, the amount of fermentation medium usedcan comprise from about 2 L to about 75 L of substantially APFfermentation medium, preferably from about 2 L to about 30 L ofsubstantially APF fermentation medium. As an example, from about 100 mgto about 5 grams, preferably from about 100 mg to about 3 grams, morepreferably from about 100 mg to about 1 gram of the biologically activebotulinum neurotoxin is obtained from the process. As an example, fromabout 20 mg to about 100 mg or from about 20 mg to about 80 mg of thebiologically active neurotoxin may be obtained per liter of thefermentation medium used. Fermentation medium can comprise vegetablederived protein product, yeast extract and glucose, for example. As anexample, the fermentation medium comprises about 5% w/v or less of avegetable derived protein product. In yet another example, thefermentation medium comprises about 2% w/v or less of a yeast extract.In a further embodiment, the fermentation medium comprises about 2% w/vor less of glucose. In a particular example, fermentation mediumcomprises about 5% w/v or less of a vegetable-derived protein product,about 2% w/v or less of a yeast extract and about 2% w/v or less ofglucose, the vegetable-derived protein product, yeast extract andglucose being in any ratio in accordance with the recited w/v percentageamounts. In some embodiments, the fermenting step proceeds for betweenabout 60 hours to about 80 hours.

In one embodiment, a substantially APF process utilizing chromatographyfor obtaining a biologically active botulinum neurotoxin, the processcomprising the following sequential steps, is provided where culturingClostridium botulinum bacteria in a substantially APF culture medium,then fermenting Clostridium botulinum bacteria from the culture mediumin about 2 L to about 75 L of a substantially APF fermentation medium,more preferably in about 2 L to about 30 L of a substantially APFfermentation medium, where at least one of the substantially APF culturemedium and substantially APF fermentation medium include a vegetableprotein, followed by harvesting the fermentation medium by removingcellular debris present in the fermentation medium and concentrating theharvested fermentation medium by filtration, and diluting theconcentrated fermentation medium by adding a buffer. Once buffered, afirst contacting step is executed, in which the diluted harvestedfermentation medium is contacted with an anion exchange media so thatthe biologically active botulinum neurotoxin is associated with theanion exchange media, then eluting the captured botulinum neurotoxinfrom the anion exchange media proceeds to thereby obtain a first eluent,followed by a second contacting step in which the first eluent iscontacted with a cation exchange media to remove impurities from thefirst eluent, thereby obtaining a second eluent; which is thenprocessed, such as by UF and/or DF; and then filtering the processedsecond eluent, thereby obtaining biologically active botulinumneurotoxin using a substantially APF process that utilizeschromatography.

As an example, the time for completion of the process, from culturingthe bacteria to obtaining the biologically active botulinum neurotoxincan be from between about 50 hours to about 150 hours, more preferablyabout 80 hours to about 120 hours, for example. In particularembodiments, the culture medium comprises no more than about 4% w/v of avegetable-derived protein product, in another, the culture mediumcomprises no more than about 2% w/v of a yeast extract and yet in stillanother, the culture medium comprises no more than about 2% w/v glucose.The culture medium can comprise the vegetable-derived protein product,yeast extract and glucose in any ratio in accordance with the recitedw/v percentage amounts. In a specific example, the pH level of theculture medium can be from about pH 6.5 to about pH 8.0, preferablyabout pH 6.8 to about pH 7.6, more preferably 7.3 at the commencement ofthe culturing step. The culturing step can be carried out for betweenabout 8 hours and about 14 hours, about 10 hours to 12 hours, preferablyabout 11 hours, at a temperature of from about 33° C. to about 37° C.,preferably at about 34.5° C., in an anaerobic chamber. In a particularexample, the anaerobic chamber can contain an integral high efficiencyparticular filter within its workspace, where culturing is conducted.The fermenting step can be carried out for between about 60 hours andabout 80 hours, preferably about 72 hours at a temperature of from about33° C. to about 37° C., preferably at 35° C. In accordance with oneaspect of our invention, the harvesting step can remove at least about80% of RNA and DNA contained in the fermentation medium and the anionexchange media can remove all measurable remaining DNA and RNA (belowlimit of detection) in the harvested fermentation medium. In anotheraspect, the harvesting step can be carried out for between about 1 hourand about 3 hours, preferably about 2.5 hours. In particular examples,the harvesting step can be carried out until 75% of the originalfermentation medium volume has been collected. In one aspect of anembodiment, the concentrating step can be carried out for between about30 minutes and about 2 hours, preferably about 0.75 hour. In anotheraspect, the diluting step dilutes the harvested fermentation medium backup to the initial weight of the fermentation medium at the commencementof the harvesting step. A first contacting step can be carried out forbetween about 4 hours and about 5 hours, for example. In one example,the first eluate from the anion exchange resin is collected atspectrophotometer readings of from about 150 mAU or greater, untilspectrophotometer readings at 280 nm decrease from peak apex back toabout 150 mAU. The second contacting step can be carried out for between1 hour and about 3 hours, preferably for about 2 hours. This secondeluate can be collected from the cation exchange resin atspectrophotometer readings from about 100 mAU or greater, untilspectrophotometer readings decrease from peak apex to about 100 mAU, forexample. A step of processing this second eluent by concentration anddiafiltration can be carried out for between about 1 hour and about 2hours, preferably for about 1.5 hours. In a particular embodiment, thefiltering step includes bioburden reduction by passing the second eluentthrough a bioburden reduction filter. The bioburden reduction filter canhave a pore size of from about 0.1 μm to about 0.3 μm, preferably 0.2μm. In particular embodiments, the process can further comprise a thirdcontacting step after the second contacting step, by contacting thesecond eluent to a hydrophobic interaction media to further removeimpurities from the second eluent and to thereby obtain a third eluent.This third contacting step can be carried out for between about 1 hourand 3 hours, preferably for about 2 hours. The third eluate can becollected from the hydrophobic interaction media at spectrophotometerreadings from about 50 mAU or greater, until spectrophotometer readingsdecrease from the peak apex back to about 50 mAU, for example. Wherethere is a third contacting step, the step of processing byconcentration and diafiltration is applied to the third eluent and iscarried out for between about 2 hours and about 4 hours. Bioburdenreduction by passing the eluent that is concentrated and diafiltered(either from a 2 or 3-column process utilized) through a bioburdenreduction filter can accordingly be performed. In particularembodiments, the process further comprises a step of freezing thebiologically active botulinum neurotoxin obtained.

In particular embodiments, the substantially APF culture mediumcomprises a volume of between about 100 mL and about 500 mL. Particularculturing steps are initiated by introducing between about 100 mL andabout 500 mL of a Clostridium botulinum-containing APF working cell bankmedia to the substantially APF culture medium. The culturing step canthen take place in an anaerobic chamber for at least about 8 hours,preferably about 11 hours, at a temperature of about 34.5° C.±1° C., forexample. In one example, the working cell bank media can have a viablecell count assay of at least about 1×10⁴ colony forming units/mL ofworking cell bank media, for example about 1×10⁵ to about 5×10⁷ colonyforming units/mL of working cell bank media, and the Clostridiumbotulinum bacterium in the working cell bank can have been selected tohave a substantially uniform morphology.

In one embodiment, the working cell bank media includes about 20% byvolume glycerol, such as sterile glycerol, for example. The working cellbank media can be made by (a) growing Clostridium botulinum bacterium inan APF medium containing about 2% w/v soy peptone, about 1% w/v yeastextract, and about 1% w/v glucose in an anaerobic chamber, at atemperature of about 34.5° C.±1° C. until an optical density of analiquot of the medium measured at a wavelength of about 540 nm is about2.5±1.0 AU, and; adding glycerol to obtain a concentration of glycerolin the medium of about 20%, thereby obtaining a working cell bank. Astorage form of the working cell bank can be prepared by freezing theworking cell bank at about below −135° C., for example. The storage formof the working cell bank, for use in an exemplary process in accordancewith the present disclosure, can be thawed at ambient temperature andused to initiate the culturing step.

The culturing step can be carried out for between about 8 hours andabout 14 hours, preferably about 11 hours at a temperature of from about33° C. to about 37° C., preferably at about 34.5° C., in an anaerobicchamber, such as, for example an anaerobic chamber/cabinet having anintegrated high efficiency particulate air (HEPA) filter, preferablywithin its workspace. The fermenting step can be carried out for betweenabout 20 hours and about 80 hours, preferably from about 60 hours toabout 80 hours, more preferably for about 72 hours at a temperature offrom about 33° C. to about 37° C., preferably at 35° C. The process canfurther comprise, for example and before the culturing step, a step ofallowing for oxidative reduction of the substantially APF culture mediumby exposing the medium to the atmosphere of an anaerobic chamber. Theprocess can also include before the fermenting step, a step of allowingfor oxidative reduction of the substantially APF fermentation medium byalso exposing the fermentation medium to the atmosphere of an anaerobicchamber. As one example, the step of allowing for oxidative reduction ofthe substantially APF culture medium can be carried out for betweenabout 10 hours and about 14 hours in the anaerobic chamber. Similarly,the step of allowing for oxidative reduction of the substantially APFfermentation medium in the fermentor can be carried out for betweenabout 10 hours and about 14 hours before the beginning of the fermentingstep. In one embodiment, an APF process, including chromatography, forobtaining a biologically active botulinum neurotoxin is disclosed,comprising the following sequential steps of adding Clostridiumbotulinum bacteria from an APF working cell bank to an APF culturemedium; culturing the Clostridium botulinum bacteria in the culturemedium; fermenting Clostridium botulinum bacteria from the culturingstep in an APF fermentation medium until Clostridium botulinum celllysis occurs; harvesting the APF fermentation culture to provide aharvested fermentation medium; subjecting the harvested fermentationmedium to concentration by filtration; diluting the filteredfermentation medium by addition of a buffer to obtain a dilutedfermentation medium; a first contacting step in which the dilutedfermentation medium is contacted with a capture chromatography media,wherein the capture chromatography media is an anion exchange media; asecond contacting step wherein an eluent from the first contacting stepis contacted with a polishing chromatography media, wherein thepolishing chromatography media is a cation exchange media, and filteringthe eluent from the second contacting step, thereby obtainingbiologically active botulinum neurotoxin by the improved APF process. Inparticular embodiments, the process can further comprise the step ofconducting a third contacting step, after the second contacting step andbefore the filtering step, by contacting eluent from the secondcontacting step with a hydrophobic interaction media. The Clostridiumbotulinum lysis phase can occur between about 35 hours and about 70hours after commencement of the fermenting step, for example. Thefermentation medium can have a volume of between about 2 L and about 75L, between about 2 L and about 30 L, or between about 2 L and 20 L offermentation medium, for example. The whole of this process can becarried out for between about 50 hours to about 150 hours, morepreferably from about 80 hours to about 120 hours. The biologicallyactive botulinum neurotoxin thus obtained by this process can have apotency of about 2.4×10⁷ to about 5.9×10⁷ units/mg of biologicallyactive botulinum neurotoxin, for example.

In accordance with one aspect, at the end of fermentation, from about 40mg to about 85 mg of botulinum neurotoxin per liter of fermentationmedium can be obtained. Subsequent to various stages of processing(filtration/chromatography/filtration runs), from about 30 mg to about60 mg of botulinum neurotoxin per liter of fermentation medium; fromabout 5 mg to about 25 mg of botulinum neurotoxin per liter offermentation medium; from about 6 mg to about 20 mg of botulinumneurotoxin per liter of fermentation medium can be obtained.

As one embodiment, the pH of the fermentation medium can be adjusted tobe between about pH 6.0 and about pH 8, preferably between about pH 6.8and about pH 7.6 at commencement of the fermenting step, more preferablyabout pH 7.3. As another example, substantially APF chromatographicprocess for obtaining a biologically active botulinum neurotoxin is alsoprovided, the process comprising the steps of obtaining a substantiallyAPF fermentation medium containing a botulinum neurotoxin; contactingthe medium with an anion exchange chromatography resin to provide apurified eluent containing a botulinum neurotoxin; contacting the eluentwith an cation exchange chromatography resin to thereby obtain a furtherpurified eluent, and filtering the further purified eluent to therebyobtain a biologically active botulinum neurotoxin purified from asubstantially APF chromatographic process. In particular configurations,an anion chromatography column can be utilized which contains from about600 mL to about 800 mL of anion exchange chromatography resin. The anionchromatography column can have a diameter of about 8 cm to about 10 cmand an anion exchange chromatography resin bed height in the column offrom about 9 cm to about 16 cm, for example. A flow rate of fermentationmedium through the anion exchange chromatography resin can be from about140 cm/hour to about 250 cm/hour, or from about 150 cm/hour to about 160cm/hour, for example. In another aspect, from about 150 mL to about 300mL of cation exchange chromatography resin in a chromatography columncan be utilized in the process, where the cation chromatography columnhas a diameter of about 5 cm to about 8 cm and a cation exchangechromatography resin bed height of from about 5 cm to about 11 cm, forexample. The process can include at least one of a diafiltration stepand/or a bioburden reduction step. The bioburden reduction step canutilize a capsule filter. The diafiltration of purified eluent ispreferably performed before a bioburden reduction step. In one example,the step of diafiltering the further purified eluent is either precededor followed by adjusting the concentration of the diafiltered furtherpurified eluent, and passing the concentration-adjusted diafilteredfurther-purified eluent through a bioburden reduction filter. Theprocess can provide a botulinum neurotoxin obtained having potency, asdetermined by a mouse LD50 bioassay, of from at least about 2.0×10⁷units/mg of botulinum toxin, such as about 2.4×10⁷ to about 6.0×10⁷units/mg of botulinum neurotoxin. Exemplary recovery at the end of theprocess of from about 4 mg to about 25 mg of botulinum toxin can berecovered per liter of fermentation media, for example.

In another embodiment, an essentially APF process for purifying abiologically active botulinum neurotoxin can comprise the steps ofobtaining from about 2 L to about 30 L an APF fermentation medium thatincludes a botulinum neurotoxin; harvesting the APF fermentation mediumstep to provide a harvested APF fermentation medium; performing anionexchange chromatography upon the harvested APF fermentation medium tothereby provide a first eluent; contacting the eluent from the anionexchange chromatography with cation exchange chromatography media toperform cation exchange chromatography to thereby provide a secondeluent; and filtering the second eluent from the cation exchangechromatography media, thereby obtaining a purified botulinum neurotoxin,wherein the purified botulinum neurotoxin obtained has a potency of fromabout 2.4×10⁷ to about 5.9×10⁷ units/mg of biologically active botulinumneurotoxin and can be obtained in a quantity of between about 4 mg toabout 25 mg per liter of APF fermentation medium used.

Our invention also comprises a compounding method for making asubstantially APF pharmaceutical composition in which the activeingredient is a biologically active botulinum neurotoxin, comprising thesteps of obtaining a biologically active botulinum neurotoxin by (i)providing a fermentation medium which is substantially free of animalproducts; (ii) fermenting Clostridium botulinum bacteria in thefermentation medium, and (iii) recovering the biologically activebotulinum neurotoxin from the fermentation medium, using an anionexchange chromatography media followed by use of a cation exchangechromatography media; and then compounding the botulinum neurotoxin withat least one suitable excipient to thereby making a substantially APFpharmaceutical composition. In one example, the method includes the stepof drying the compounded botulinum neurotoxin and at least one suitableexcipient to obtain a stable form for shipment or storage, by freezedrying or lyophilization or vacuum drying, in which the activeingredient is the biologically active botulinum neurotoxin, where thefermentation medium comprises a protein product obtained from avegetable. The vegetable from which the protein product can obtained canbe a soy, corn or malt, debittered seed of Lupinus campestris, orhydrolyzed products therefrom. The botulinum neurotoxin obtained canhave a potency between about 2.0×10⁷ units/mg of botulinum neurotoxin toabout 6.0×10⁷ units/mg of botulinum neurotoxin. The botulinum neurotoxinis selected from the group consisting of botulinum neurotoxins types A,B, C₁, D, E, F and G, preferably botulinum neurotoxin type A. Inparticular instances the botulinum neurotoxin is obtained as a botulinumtoxin neurotoxic component with a molecular weight of about 150 kDa freeof the complexing proteins of a botulinum toxin complex. In particularembodiments, the suitable excipient is selected from the groupconsisting of albumin, human serum albumin, recombinant human serumalbumin, gelatin, sucrose, trehalose, hydroxyethyl starch, collagen,lactose, sucrose, amino acid, sodium chloride, potassium chloride,polysaccharide, caprylate, polyvinylpyrrolidone and potassium citrate.Obtaining the biologically active botulinum neurotoxin can furthercomprise the step of using a hydrophobic interaction media following useof the cation exchange media. In particular examples, vacuum dryingtakes place at a temperature of about 20° C. to about 25° C. In someembodiments, the vacuum drying takes place at a pressure of about 70mmHg to about 90 mmHg, for example. The time for vacuum drying can befrom about 4 hours to about 5 hours, for example.

Particular aspects of the present disclosure are directed to providing apharmaceutical composition, which can, for example, comprise abiologically active botulinum neurotoxin complex and an excipient isselected from the group consisting of albumin, human serum albumin,recombinant human serum albumin, gelatin, sucrose, trehalose,hydroxyethyl starch, collagen, lactose and sucrose, where thepharmaceutical composition is essentially free of nucleic acid.

In particular examples, a pharmaceutical composition is provided thatcomprises a biologically active botulinum neurotoxin wherein thebotulinum neurotoxin obtained has a potency between about 2.0×10⁷ toabout 6.0×10⁷ units/mg of biologically active botulinum neurotoxin andat least one excipient, where the composition comprises less than about12 ppm of nucleic acid, preferably less than 1 ppm of nucleic acid permg of botulinum neurotoxin complex.

In a particular embodiment, a substantially APF chromatographic processfor obtaining a biologically active botulinum neurotoxin comprises thefollowing sequential steps of: culturing Clostridium botulinum bacteriain a substantially APF culture medium for between about 10 hours andabout 12 hours, or until a biomass measurement of culture medium has anoptical density, at a wavelength of about 540 nanometers (nm), ofbetween about 0.8 AU and about 4.5 AU; fermenting Clostridium botulinumbacteria from the culture medium in a substantially APF fermentationmedium for between about 65 hours to about 75 hours or until a biomassmeasurement is taken at the end of fermentation by measuring the opticaldensity of the fermentation medium using a online biomass probe at awavelength of about 890 nm is between about 0.05 AU and about 0.7 AU;harvesting the fermentation medium for about 2.5 hours, whereby cellulardebris in the fermentation medium is removed and the weight offermentation medium is reduced to about three quarters of its startingweight at the beginning of the harvesting step; concentrating theharvested fermentation medium by tangential flow filtration to about onequarter of its starting volume at the beginning of the harvesting step;diluting the concentrated fermentation medium by adding a buffer,wherein the concentrating and diluting steps take place for betweenabout 0.5 hour to about 2 hours, whereby during concentration thefermentation medium is reduced to about one quarter of its startingweight at the beginning of the harvesting step, and is then diluted, bythe addition of the buffer, back up to its original starting weight atthe beginning of the harvesting step; contacting the dilutedfermentation medium with a capture chromatography media, to capture thebiologically active botulinum neurotoxin, for a time period of about 4hours to about 5 hours; contacting eluent from the capturechromatography media with a first polishing chromatography media toconduct a first polishing run to remove impurities therefrom for a timeperiod of about 1.5 hours to about 2.5 hours; conducting a secondpolishing run by passing eluent from the polishing chromatography mediathrough a hydrophobic interaction media for a time period of about 1.5hours to about 2.5 hours; processing eluent from the hydrophobicinteraction media by diafiltration, for a time period of about 1 hour toabout 4 hours; and filtering the processed eluent through a bioburdenreduction filter, for about 0.5 hour, thereby obtaining biologicallyactive botulinum neurotoxin.

In another aspect, a substantially APF chromatographic system forobtaining a biologically active botulinum neurotoxin is disclosed, thesystem comprising: a first apparatus for culturing Clostridium botulinumbacteria, the first apparatus capable of containing a substantially APFculture medium; a second apparatus for fermenting Clostridium botulinumbacteria which have been cultured in the first apparatus, the secondapparatus capable of containing a substantially APF fermentation medium;a third apparatus for harvesting the fermentation medium; a fourthapparatus for carrying out concentrating and diluting the harvestedmedium from the third apparatus; the fourth apparatus comprisingtangential flow filtration (TFF); a fifth apparatus for carrying out afirst purification of the botulinum neurotoxin from the concentrated anddiluted medium, the fifth apparatus comprising an anion exchangechromatography media, thereby obtaining a first purified botulinumneurotoxin; and a sixth apparatus for carrying out a second purificationof the first purified botulinum neurotoxin, the sixth apparatuscomprising a cation exchange chromatography media, and thereby obtaininga second purified botulinum neurotoxin.

In a particular embodiment, the system can further comprise a seventhapparatus for carrying out a further purification, by purifying thesecond purified botulinum neurotoxin obtained from the sixth apparatus,wherein the seventh apparatus comprises a hydrophobic interaction media,thereby obtaining a third purified botulinum neurotoxin. The system canalso further be comprised of an eighth apparatus having a filtrationmembrane for filtering eluent from the sixth or seventh apparatus.

In still yet another embodiment, a chromatography column with a diameterof between about 8 cm and about 15 cm contains the anion exchangechromatography media and the anion exchange chromatography media canhave a bed height in the column of between about 8 cm and about 15 cm,for example. In still another example, the system's fourth apparatuscomprises a chromatography column that is operated at a flow rate ofbetween about 125 cm/hour and about 200 cm/hour, and the column can havea column volume between about 500 mL and about 1 L. In one aspect, thefifth apparatus can have a column volume of from about 50 mL and about500 mL, and a bed height of from about 8 cm and about 15 cm, forexample. In some examples, the fifth apparatus' chromatography columnhas a column diameter from about 2 cm and about 10 cm, for example. Thefifth apparatus' chromatography column can have an exemplary flow rateof between about 100 cm and about 200 cm/hour. The seventh apparatus ofthe system can comprise a filtration membrane.

In another embodiment, the system can further comprise a ninthapparatus, the ninth apparatus comprising an anaerobic chamber forproviding an anaerobic atmosphere where the first apparatus forculturing Clostridium botulinum bacteria is contained therein. Thisninth apparatus preferably includes an integrated high efficiencyparticulate air (HEPA) filter located within its chamber/workstation.The second apparatus of the system (for fermentation) can include atleast one probe for detecting oxidation-reduction potential or pH oroptical density. In a particular example, an at least one disposableprobe is selected from the group consisting of a reduction-oxidationprobe, a pH probe and a turbidity probe. A eighth apparatus of thesystem can comprise a tangential flow filtration apparatus forconcentration and buffer exchange. In a further embodiment, the systemcan comprise an tenth apparatus that includes a bioburden reductionapparatus for reducing bioburden. In one example, the bioburdenreduction apparatus comprises a filter having a pore size of aboutbetween about 0.1 μm and 0.3 μm, preferably 0.2 μm. The system can alsoinclude a eleventh apparatus for use after obtaining the second purifiedbotulinum neurotoxin, for storing the purified botulinum neurotoxin. Inone example, this storage apparatus provides a storage temperaturebetween about −25° C. to about −80° C.

In another aspect, a biologically active botulinum toxin is provided byan APF process having the following steps of providing a substantiallyAPF fermentation medium; fermenting a Clostridium botulinum bacteria inthe fermentation medium; recovering the biologically active botulinumneurotoxin from the fermentation medium using an anion exchangechromatography media followed by use of a cation exchange chromatographymedia, where the biologically active botulinum toxin obtained has apotency between about 2.0×10⁷ units/mg of botulinum neurotoxin to about6.0×10⁷ units/mg of botulinum neurotoxin. In one embodiment, the processfurther comprises the step of further purifying the botulinum neurotoxinby using a hydrophobic interaction media following use of the cationexchange media.

In accordance with another aspect, a method for treating a condition ina patient is provided, utilizing a pharmaceutical composition comprisingthe botulinum neurotoxin obtained in accordance with the methods hereindisclosed. A condition can include a disease, ailment, sickness, orcosmetic deformity or appearance. In one example, the method of treatinga condition in a patient comprises the step of administering to thepatient a therapeutically effective amount of a pharmaceuticalcomposition comprising a botulinum neurotoxin and at least one suitableexcipient, where the botulinum toxin has a potency of about 1 unit about0.02 picograms to thereby treat the condition of the patient.

In a particular example, the botulinum neurotoxin for treating theseconditions can be obtained by a process of culturing Clostridiumbotulinum bacteria in a substantially APF culture medium; obtaining asubstantially APF fermentation medium containing the botulinumneurotoxin; contacting the medium with an anion exchange chromatographymedia to provide a purified eluent containing the botulinum neurotoxin;contacting the eluent with an cation exchange chromatography media tothereby obtain a further purified eluent, and filtering the furtherpurified eluent to thereby obtain the biologically active botulinumneurotoxin purified from a substantially APF chromatographic process.

In one embodiment a substantially APF chromatographic system forobtaining a biologically active botulinum neurotoxin is included, thesystem comprising a first apparatus for anaerobic culturing Clostridiumbotulinum bacteria, the first apparatus capable of containing from about200 mL to about 1 L of a substantially APF culture medium; a secondapparatus for anaerobic fermentation of Clostridium botulinum bacteriawhich has been cultured in the first apparatus, the second apparatuscapable of containing from about 5 L to about 75 L, or from about 2 L toabout 75 L, or from about 2 L to about 30 L of a substantially APFfermentation medium and including at least one disposable probe selectedfrom the group consisting of a reduction-oxidation probe, a pH probe anda turbidity probe; a ninth apparatus for providing an anaerobicatmosphere and capable of containing the first apparatus, the ninthapparatus comprising an anaerobic chamber having an integrated highefficiency particulate air filter within the chamber, wherein saidchamber can contain the first apparatus for anaerobic culturingClostridium botulinum bacteria; a third apparatus for harvesting thefermentation medium; a fourth apparatus for carrying out concentrationand dilution of the harvested medium, a fifth apparatus for carrying outa first purification of botulinum neurotoxin obtained from the fourthapparatus, the fifth apparatus comprising an anion exchangechromatography media, thereby obtaining a first purified botulinumneurotoxin; a sixth apparatus for carrying out a second purification ofthe first purified botulinum neurotoxin, the sixth apparatus comprisinga cation exchange chromatography media, thereby obtaining a secondpurified botulinum neurotoxin; a seventh apparatus carrying out a thirdpurification of the second purified botulinum neurotoxin, the seventhapparatus comprising hydrophobic interaction media, thereby obtaining athird purified botulinum neurotoxin; and an eighth apparatus forconcentration and buffer exchange of the third purified botulinumneurotoxin, the eighth apparatus comprising a TFF membrane.

In particular examples, the fermentation medium comprises no more thanabout 5% w/v of a vegetable-derived protein product, no more than about2% w/v of a yeast extract and no more than about 2% w/v glucose, andwhere the pH level of the fermentation medium is from about pH 6.8 toabout 7.6, preferably about pH 7.3 at the start of an about 72 hourfermenting step, for example. In one embodiment, the method can furthercomprise the step of contacting the further purified eluent with ahydrophobic interaction media to obtain an even further purified eluentcontaining the botulinum neurotoxin. In a particular example, the methodof treating the conditions can be by using a botulinum neurotoxin thatis obtained as a botulinum toxin neurotoxic component with a molecularweight of about 150 kDa free of the complexing proteins of a botulinumtoxin complex. Exemplary administration steps can be selected from thegroup of administration routes consisting of intramuscular, intradermal,subcutaneous, intraglandular, intrathecal, rectal, oral and transdermaladministration, and the botulinum neurotoxin is selected from the groupconsisting of botulinum toxin type A, B, C₁, D, E, F or G. Preferably,the botulinum neurotoxin is botulinum neurotoxin type A.

In some examples, the system can facilitate a process whereby abiologically active botulinum neurotoxin complex can be obtained for useas part of pharmaceutical composition that comprises less than about 12ng of nucleic acid per mg of botulinum neurotoxin complex, preferablybelow 1 ng of nucleic acid per mg of botulinum neurotoxin complex, morepreferably having no measurable a nucleic acid (e.g. below a limit ofdetection).

DRAWING

FIG. 1A is a flow chart showing major steps in the Example 1 NAPFprocess. FIG. 1B is a flow chart showing major steps in the Example 2IAPF process, wherein the capture and polishing chromatography steps canutilize either a 2-columns (anion exchange followed by cation exchange)or 3-columns (FAPF) (anion exchange followed by cation exchange followedby a hydrophobic interaction column).

DESCRIPTION

Our invention is based on the discovery that a high potency, high puritybiologically active Clostridial neurotoxin, such as a botulinumneurotoxin, can be obtained by use of a simple, fast and economical APFchromatographic system and process. Significantly, use of our system andprocess can result in a purified botulinum neurotoxin comprising 1 ng(or less than 1 ng) of nucleic acid (RNA and DNA) impurities per 1 mg ofthe purified botulinum neurotoxin obtained, even though no animalderived enzymes, such as RNase and DNase, are used to purify thefermented botulinum neurotoxin. For example, use of our system andprocess can result in a purified botulinum neurotoxin comprising lessthan about 0.6 ng of nucleic acid (RNA and DNA) impurities per milligramof purified botulinum neurotoxin, obtained. The botulinum neurotoxinobtained can be a botulinum toxin type A complex, such as a 300 kDa, 500kDa or 900 kDa (approximate molecular weights) complex or mixturesthereof. The botulinum neurotoxin obtained can also be a botulinum toxintype neurotoxic component (i.e. without the complex proteins) with amolecular weight of about 150 kDa. The botulinum neurotoxin can be anyone of the serotypes A, B, C, D, E, F or G or mixtures thereof.Additionally, the improved systems and processes can be practiced inconjunction with a recombinant, hybrid, chimeric or modified botulinumtoxin (light chain, heavy chain, or both chains together).

An important aspect of our invention is use of an anion exchange(capture) media chromatography followed by use of cation exchange(polishing) media chromatography to purify botulinum neurotoxin from anAPF fermentation medium in which Clostridium botulinum bacterium havebeen fermented. We found that use of anion exchange followed by use ofcation exchange chromatography media provides an effective and rapidmethod for obtaining high purity, high yield botulinum neurotoxin.Previously, it had been thought that use of anion exchangechromatography has a detrimental effect on gel banding patterns ofbotulinum neurotoxin, thereby discouraging use of anion exchangechromatography for botulinum neurotoxin purification. See e.g. U.S. Pat.No. 7,452,697 at column 55, lines 53-57.

Another important aspect of our invention is that it results in highpurity botulinum neurotoxin (i.e. 1 ng nucleic acid/mg botulinumneurotoxin obtained), as set forth above. A further important aspect ofour invention is that whereas the known Schantz process requires severalweeks (i.e. typically about 18 to about 22 days) to culture, ferment andpurify the botulinum neurotoxin, a system and process within the scopeof our invention permits all culturing, fermentation and purificationsteps to be completed in one week or less. In a preferred embodiment ofour invention all culturing, fermentation and purification steps can becompleted in six days or less. In a more preferred embodiment of ourinvention all culturing, fermentation and purification steps can becompleted in about four days or less (e.g. within about 80 to about 144hours or within a time/range therebetween). We invented this rapid, moreembodiment of our invention by developing an eight or nine step process(and the system for accomplishing the process) and by finding that eachof the eight or nine steps in a particular embodiment can be completedwithin the time periods set forth below:

about 8 hours to about 14 hours for culturing;about 60 hours to about 80 hours for fermenting;about 2.5 hours for harvesting;about 2 hours to about 4 hours for concentrating and diluting;about 4 hours to about 6 hours for anion exchange chromatography (thisincludes time for eluting captured botulinum toxin)about 2 hours for cation exchange chromatography;about 2 hours for an optional third chromatography step (i.e.hydrophobic interaction chromatography;about 2 hours to about 4 hours for concentration and diafiltration, and;about ½ hour for further filtration. Thus, the total time required tocomplete our 8 or 9 step rapid, more preferred embodiment of ourinvention is from about 75 hours to about 150 hours.

Our invention is more efficient and time saving. In one aspect, our newprocess utilizes pre-selected and verified cell lines, and thus doesaway with the prior art Shantz process steps of plating and growingcells, selecting and harvesting colonies, and step-up cell-lineexpansion of the harvested colonies (prior to cell culturing andfermentation steps) that were needed to culture and then inoculatefermentation medium. In one aspect, our invention begins straight awaywith culturing pre-selected cells for inoculation of an APF culturemedium, thus saving time and process steps.

Through experimentation we developed two chromatography column (“IAPF”)and three column (“FAPF”/“FIAPF”) chromatography systems and processesfor purifying the botulinum neurotoxin present in the fermentationmedium, the fermentation medium resulting from an APF fermentation ofClostridium botulinum bacterium. Significantly, while an APFfermentation process can reduce or eliminate animal derived products(such as casein and meat broth) as nutrients from the media used toculture and ferment Clostridial bacteria, known APF fermentationprocesses are typically followed by one or more purification steps whichmake use of animal derived products, such as the enzymes DNase andRNase. Our systems and processes for purifying the botulinum neurotoxinpresent in an APF fermentation medium do not use animal derived enzymes.

Our invention can encompass loading a harvested fermentation medium(e.g. clarified by filtration) onto an anion exchange column such as aPOROS® 50HQ anion exchange chromatography resin from Applied Biosystems.In one aspect, a strong anion exchange media can be used, having a basematrix of polystyrene/divinylbenzene and particle diameter of about 50μm and dynamic capacity (BSA mg/ml) of about 60-70. The anion exchangecolumn captures the Clostridial neurotoxin (such as a botulinum toxincomplex) and reduces impurity levels. It was found that an anionexchange column provided an efficient capture of a botulinum toxincomplex from harvested fermentation medium with retention of thebiological activity of the botulinum toxin complex, while alsoseparating many impurities present with the botulinum toxin in thefermentation medium. A suitable buffer is used to elute the captured(bound) Clostridial neurotoxin from the anion exchange column.

In a two-column embodiment of our invention, eluent (containing thebotulinum neurotoxin) from the anion exchange column is loaded onto acation exchange column to further purify the botulinum neurotoxin fromimpurities. The cation exchange column can be a POROS® 20HS cationexchange resin from Applied Biosystems. In one aspect, a strong cationexchange media can be used, having a base matrix ofpolystyrene/divinylbenzene and particle diameter of about 20 μm anddynamic binding capacity (lysosyme mg/ml) of about >75. In athree-column embodiment (FAPF) of our invention, eluent from the cationexchange column is loaded onto a hydrophobic interaction column such asPhenyl Sepharose HP resin from GE Healthcare to further purify thebotulinum neurotoxin. In one aspect, a matrix of highly cross-linkedagarose beads with a particle size of about 34 μm, which have beenderivitized with phenyl groups and have a dynamic binding capacity(chymotrypsinogen mg/ml) of about 45, may be used.

After either the two column or three column process, eluent from thelast used column can be further processed to obtain highly purified bulkbotulinum toxin complex. Post-chromatography processing steps caninclude concentration and buffer exchange by ultrafiltration anddiafiltration, sterile filtration and preparation of a solution ofpurified botulinum toxin complex instead of a suspension (prior art),preferably in potassium citrate, and in one example, at a concentrationof 10 mM potassium citrate at a pH of about 6.5.

In certain preferred embodiments, the media for the growth (anaerobicculturing and anaerobic fermentation) of Clostridium botulinum andproduction of botulinum toxin can comprise soy based products to replaceanimal derived products so that media used are substantially or entirelyfree of animal-derived products. The culture step increases the quantityof microorganism for subsequent fermentation. Culturing permits dormant,previously frozen bacteria to rejuvenate into actively growing cultures.Additionally, the volume and quantity of viable microorganisms used toinoculate the fermentation medium can be controlled more accurately froman actively growing culture than it can be from a stored,non-propagating Clostridium botulinum cell bank. Thus, a sample of aworking cell bank in APF media is thawed and placed in the selected APFculture medium. Upon obtaining a suitable level of bacterial growth theculture medium is used to inoculate the fermentation medium. As oneexample, from about 1% to about 5%, or an amount therebetween, of theculture medium having Clostridium botulinum from the growth phase isused to inoculate the fermentation medium. Fermentation is carried outto produce the maximum amount of microbial cells in a large-scaleanaerobic environment (Ljungdahl et al., Manual of industrialmicrobiology and biotechnology (1986), edited by Demain et al, AmericanSociety for Microbiology, Washington, D.C. page. 84). Alternately,growth of Clostridium botulinum in the fermentation medium can proceedby adding the sample of the working cell bank directly to thefermentation medium.

In the prior art, growth of Clostridium botulinum in the culture mediumtypically proceeds in two stages, a first stage of cell plating, cellcolony growth, selection and growth, followed by a second stage ofinoculation of culture medium (typically a two stage step-up culture)and inoculation of fermentation medium and botulinum toxin production.Preferably, growth in the culture media in any stage does not result incell lysis before inoculation of fermentation media with the finalgrowth in culture medium. Thus, prior to our invention it took aboutfour days to culture Clostridium botulinum bacteria before thefermentation step was begun. In accordance with our invention we areable complete all culturing in only 8 to 14 hours because there is noneed for the previously utilized steps of plating cells, subsequentwaiting time for colony growth on blood agar plates, selection ofcolonies from the plates for growth in small volumes of culture (e.g.8-9 mL) that then provide an inoculum for the culturing medium. Inaccordance with one aspect of our invention, pre-selected cells aredirectly utilized to inoculate the culture medium that is then utilizedto inoculate the full-scale fermentation medium from which botulinumtoxin is eventually purified, thus eliminating the plating, colonyformation, selection and step up steps previously utilized to grow cellsthat would inoculate a culture medium which is then itself utilized toinoculate fermentation medium.

Animal-based (non-APF or “NAPF”) culture media generally include brainheart infusion media (BHI), bacto-peptone, NaCl, and glucose. Culturemedia within the scope of our invention are APF culture media. Forexample, a soy-based product can be used instead of BHI andbacto-peptone in the culture and fermentation media. Preferably, thesoy-based product is soluble in water and comprises hydrolyzed soy,although Clostridium botulinum can grow in media containing insolublesoy. Any source of soy-based products may be used in accordance with thepresent invention. Preferably, the soy is hydrolyzed soy and thehydrolyzation has been carried out using non-animal enzymes. Sources ofhydrolyzed or soluble soy include Hy-Soy (Quest International), Soypeptone (Gibco) Bac-soytone (Difco), AMISOY (Quest), NZ soy (Quest), NZsoy BL4, NZ soy BL7, SE50M (DMV International Nutritionals), and SE50MK(DMV).

EXAMPLES

The following examples set forth particular embodiments of our inventionand are not intended to limit the scope of our invention. Unlessotherwise set forth in the examples “toxin” or “botulinum toxin” means abotulinum toxin type A complex with a molecular weight of about 900 kDa.Systems and method disclosed herein for purifying a botulinum toxin typeA complex with a molecular weight of about 900 kDa, have readyapplicability to the purification of about 150 kDa, about 300 kDa, about500 kDa as well as other molecular weight toxins, complexes, botulinumtoxin serotypes and botulinum toxin neurotoxic component.

Example 1 Non-APF (Schantz) Process for Obtaining a Botulinum Toxin

This example sets forth the prior art Schantz process for obtainingbotulinum neurotoxin. The process is a non-APF process using animalderived media and reagents (i.e. beef blood agar plates for culturing,casein in the fermentation medium and use of RNase and DNase enzymes forbotulinum neurotoxin purification). FIG. 1A is a flow chart showing themajor steps of the Schantz process. The Schantz process has about 16 to20 major steps, for production scale work uses a 115 L fermentor andtakes about 3 weeks to complete. The Schantz process is commenced bythawing a non-APF Clostridium botulinum master cell bank (MCB) vial toroom temperature followed by four cultivation steps. First to selectcolonies with a suitable morphology, aliquots from the thawed MCB vialwere streaked on pre-reduced Columbia blood agar (CBA) plates andanaerobically incubated for 30-48 hours at 34° C.±1° C. Second, selectedcolonies were inoculated into 9 mL test tubes containing a casein growthmedium for 6-12 hours at 34° C. The contents of the 9 mL tube with themost rapid growth and highest density (growth selection step) were thenfurther cultivated through two step-up anaerobic incubations (the thirdand fourth cultivation steps), being a 12-30 hour incubation at 34° C.in a 600 mL to 1 L seed cultivation bottle, followed by a cultivation ina 15 L to 25 L seed fermentor containing a casein growth medium for 6-16hours at 35° C. These two step-up cultivations were carried out in anutritive media containing 2% casein hydrolysate (a casein [milkprotein] digest), 1% yeast extract and 1% glucose (dextrose) in water atpH 7.3.

The step-up cultivations were followed by a further incubation for 60-96hours at 35° C. in a commercial scale (i.e. 115 L) production fermentorin a casein containing medium under a controlled anaerobic atmosphere.Growth of the bacterium is usually complete after 24 to 36 hours, andduring the fermentation step carried out for about 65 to about 72 hourswhere most of the cells undergo lysis and release botulinum neurotoxin.It is believed that toxin is liberated by cell lysis and activated byproteases present in the media. A filtrate of the culture medium can beprepared using a single layer depth filter to remove gross impurities(i.e. whole and ruptured cells) thereby obtaining a clear solutionreferred to as a clarified culture. Collection of botulinum neurotoxinfrom clarified culture was accomplished by lowering the pH of theclarified culture to pH 3.5 with 3M sulfuric acid to precipitate the rawtoxin at 20° C. (acidification precipitation). The raw botulinumneurotoxin was then concentrated (to achieve a volume reduction) byultramicrofiltration (microfiltration) (referred to as MF or UF)followed by diafiltration (DF). A 0.1 μm filter was used for themicrofiltration step.

The harvested crude or raw toxin was then transferred to a digestionvessel and stabilized by addition of the protease inhibitor benzamidinehydrochloride. DNase and RNase were added to digest (hydrolyze) nucleicacids. Hydrolyzed nucleic acids and low molecular weight impurities werethen removed by further UF and DF steps. The toxin was then extractedwith pH 6.0 phosphate buffer and cell debris removed by clarification.Next three sequential precipitation steps (cold ethanol, hydrochloricacid and ammonia sulfate precipitations) were carried out. The purifiedbotulinum neurotoxin complex (bulk toxin) was stored as a suspension ina sodium phosphate/ammonium sulfate buffer at 2° C. to 8° C.

Completion of this Example 1 Schantz (non-APF) process, including theharvesting and purification steps, takes about two to three weeks. Theresulting bulk botulinum neurotoxin was a high quality suspension of 900kDa botulinum toxin type A complex made from the Hall A strain ofClostridium botulinum with a specific potency of ≧2×10⁷ U/mg, anA₂₆₀/A₂₇₈ of less than 0.6 and a distinct pattern of banding on gelelectrophoresis, and suitable for use for the compounding of a botulinumtoxin pharmaceutical composition.

Botulinum neurotoxin can also be obtained from an APF,non-chromatographic process, as set forth in Example 7 of U.S. Pat. No.7,452,697, the complete APF, non-chromatographic process (from beginningof culturing to end of all purification and processing steps) takingabout two to three weeks to complete. Alternately, botulinum neurotoxincan also be obtained from an APF, chromatographic process, as set forthin Example 16 of U.S. Pat. No. 7,452,697, the APF, chromatographicprocess (from beginning of culturing to end of all purification andprocessing steps) taking a week or longer to complete.

Example 2 APF, Two and Three Column Chromatographic Systems andProcesses for Obtaining a Botulinum Neurotoxin

We developed rapid APF, anion-cation chromatographic based systems andprocesses for obtaining high yield, high purity botulinum neurotoxin.The process of this Example 2 had only 8-10 major steps, for productionpurposes (that is to obtain gram quantities of the final botulinumneurotoxin) used a 20 L fermentation vessel and takes only 4-7 days,preferably about 4 to about 6 days, to complete all step of the processfrom initiation of culturing to completion of final purification andtoxin storage. Apparatus utilized in the systems herein disclosed arediscussed below. Both a two chromatographic media process and a threechromatographic media process were developed and are set forth herein.The two media process used anion exchange chromatography followed bycation exchange chromatography. The three media process used anionexchange chromatography followed by cation exchange chromatographyfollowed by hydrophobic interaction chromatography (HIC). The HICremoved further impurities such as a 49 kDa impurity (which turns out tobe a host cell glucose phosphate isomerase, as discussed below).

Preparation of Working Cell Bank

We developed a new Clostridium botulinum cell bank (for use to initiatethe culturing step) without use of Columbia blood agar plates, and whichremoved the need for colony selection prior to cultivation and alsoeliminated the need to carry out the Shantz process step up tubecultivation and multiple seed (cultivation) steps.

For this purpose, a previously established Schantz master cell bank(MCB) was used to create an APF research cell bank (RCB) from which anew APF master cell bank (MCB) and a subsequent working cell bank (WCB)were generated. A research cell bank (RCB) was made from a colony fromthe Schantz (NAPF) MCB. To remove the animal-derived protein from theMCB vial, the cells were washed twice in APF medium containing 2% w/vSPTII (Soy Peptone type II), 1% w/v yeast extract, and 1% w/v glucose.The cells were plated on APF medium under strict anaerobic conditionsusing a Modular Atmosphere Controlled System (MACS) anaerobic chamber.An isolated colony was further expanded and stored in APF mediumcontaining about 20% glycerol below −135° C.

The APF-MCB was made under GMP conditions by expanding the RCB intooxygen-free APF medium (200 mL, reduced for a minimum of 12 hours in ananaerobic chamber) and cultured in a MACS anaerobic chamber at 34.5°C.±1° C. (stirred at 60 rpm) until the OD₅₄₀ of the culture reached2.5±1.0 AU. Sterile glycerol was added to the resultant culture to afinal concentration of about 20% after which the mixture was transferredinto cryovials at 1 mL/vial (APF-MCB vials). The vials were flash frozenin liquid nitrogen, and then stored below −135° C. An APF-WCB was madeunder GMP conditions by expanding as above. The resultant APF cell bankswere characterized for identity, purity, viability and geneticstability.

Upstream Steps (Culturing and Fermentation)

Our Example 2 process had two general stages; an upstream stage and adownstream stage. The upstream stage includes expansion of a startingcell line (growth and reproduction of Clostridium botulinum bacteria ina substantially APF culture medium), fermentation, harvest (removal ofcellular debris) to provide a clarified, harvested culture that is thenconcentrated and diluted. Thus, in this example the nine steps of ourtwo column process are culturing, fermentation, harvest filtration,concentration, capture (anion) chromatography, polishing (cation)chromatography, buffer exchange, bioburden reduction and vial fill.

The upstream stage included use of a culture medium in a 1 L bottlecontaining 400 mL of reduced (in an anaerobic chamber) seed APF culturemedium (2% w/v SPTII, 1% w/v yeast extract, (adjusted to pH 7.3 with 1 Nsodium hydroxide and/or 1 N hydrochloric acid prior to autoclaving)) 1%w/v sterile glucose added post autoclaving of culture media). Theculture (seed) medium was inoculated with 400 μL of a thawed Clostridiumbotulinum WCB. Incubation/culturing occurred at 34.5° C.±1.0° C. with150 rpm agitation in an anaerobic chamber.

When the optical density of the culture medium at 540 nm was 1.8±1.0 AU,the entire contents of the 1 L bottle (approximately 400 mL) weretransferred to a 20 L production fermentor containing APF fermentationmedium adjusted with 1 N sodium hydroxide and/or 1 N hydrochloric acidpost-steam sterilization to pH 7.3, fermentation medium composed of3.25% w/v SPTII, 1.2% w/v yeast extract, 1.5% w/v sterile glucose (addedpost sterilization; sterilization, e.g. at about 122° C. for 0.5 hour).The temperature and agitation were controlled at 35° C.±1° C. and 70rpm, respectively. Nitrogen overlay was set at 12 slpm and headspacepressure set at 5 psig to maintain an anaerobic environment for cellgrowth. Fermentation pH and cell density were monitored by pH and onlineturbidity probes, respectively. The three phases for the productionfermentation include exponential growth, stationary, and autolysisphases. Cellular autolysis, which releases active BoNT/A complex intothe culture medium, was observed to occur consistently between 35 hoursand the end of fermentation. At the end of fermentation, the culture wascooled to 25° C. for harvest.

Once the fermentation medium was cooled to 25° C., the cell debris wasseparated from the botulinum neurotoxin type A complex containing lysateby depth filtration, first through a 5-0.9 μm nominal retention ratinggradient pre-filter to remove cell debris, and then through a positivelycharged 0.8-0.2 μm nominal retention rating gradient to remove DNA(removal of up to about 80%). Both filters were rinsed together with 20L of water for injection (WFI) before use. A minimum of 15 L of thefiltrate was required for further processing, and any excess materialwas decontaminated after in-process sampling is complete. The filtratewas stored at 4° C. if not immediately processed by ultrafiltration.

Within a biosafety cabinet (BSC) the filtrate from the harvest step wasconcentrated from 15 L to 5±0.5 L using a hollow fiber, tangential flowfiltration (TFF) membrane from GE Healthcare. The ultrafiltered materialwas then diluted with 10 mM sodium phosphate pH 6.5 buffer to a finalvolume of 20 L. This material was purified by use of either 2 column(anion then cation) or three chromatography columns (anion, cation, andthen hydrophobic interaction). The diluted, ultrafiltered harvestmaterial was stored at 4° C. if not immediately processed bypurification.

In the Schantz process the culture step is ended and the fermentationstep begun based on time and visual observation of culture growth. Incontrast, in our Example 2 processes determination of when to end theculturing step is based on analysis of culture fluid optical density,which ensures that the culture is in the logarithmic growth phase at thetime of commencement of the fermentation step, and permits reduction ofduration of the culturing step to about 8 hours to about 14 hours. OurOD parameter terminated culture step maximized the health of thecultured cells and encouraged robust and abundant botulinum toxinresulting from the fermentation step. The average optical density (at540 nm) of the culture medium at conclusion of culturing was 1.8 AU. Theaverage duration of the fermentation step 72 hours and the average finalturbidity (A₈₉₀) of the fermentation medium at conclusion of thefermentation step was 0.15 AU. The average amount of botulinum toxintype A complex present (as determined by ELISA) in the 20 L fermentationmedium (whole broth) at the end of the fermentation step for was about64 μg botulinum toxin type A complex/mL fermentation medium.

The harvest step used depth filtration to remove cell debris and nucleicacids, followed by ultrafiltration and dilution to prepare thefermentation medium for the next step in the process. Thisharvesting/cell debris clearing is fundamentally different from theSchantz harvest process, which uses precipitation by acidificationfollowed by microfiltration and diafiltration to concentrate andexchange buffers in preparation for further processing.

Downstream Steps (Purification)

Downstream steps included capture of the botulinum neurotoxin on ananion exchange column, elution from the column and further separationfrom impurities by polishing on a cation exchange column, and preferably(in the three column process), passage of eluent containing desiredbotulinum neurotoxin through a third column, preferably a hydrophobicinteraction column (e.g. chromatography), followed by concentration andbuffer exchange using tangential flow filtration (TFF), and bioburdenreduction (e.g. by further filtration using a 0.2 μm filter) to a finalbotulinum neurotoxin type A complex optimized for cold storage,preferably freezing, and eventual compounding into a botulinumneurotoxin type A complex pharmaceutical composition. The sequence ofthe chromatography and filtration stages was intended to remove productand process-related impurities, to remove potential adventitious agentsand to control the botulinum neurotoxin type A complex concentration andbuffer matrix of the final botulinum neurotoxin type A in order toprovide a more stable drug substance.

A more detailed embodiment of the three column downstream processcarried out is as follows. Clarified (diluted) ultrafiltered material(20 L, as disclosed above) was passed through a POROS® 50HQ anionexchange chromatography resin, the captured botulinum neurotoxin waseluted from the anion exchange column and then run through a POROS® 20HScation exchange chromatography resin, the eluent from which was runthrough a Phenyl Sepharose HP chromatography resin. Eluent from the HICcolumn was subjected to 100 kDa tangential flow filtration, followed by0.2 μm filtration. The resulting botulinum neurotoxin type A complex wasfrozen for storage.

In this Example, we used in the first chromatography step of thedownstream process a POROS® 50HQ anion exchange chromatography resinpacked into a column with an inner diameter of about 8 cm and a columnheight of about 15 cm. The entire POROS® 50HQ column operation wascompleted at ambient temperature, and the flow was in the downwarddirection. The botulinum neurotoxin type A complex was eluted from theanion column using a pH step change where the more negatively chargedcomponents such as nucleic acids (e.g. DNAs and RNAs) and other hostcell proteins remained bound to the anion exchange column.

Particulars of the anion exchange step were: use of the POROS® 50HQcolumn using 0.1 N sodium hydroxide for a minimum contact time of 30minutes (at least about 3 column volumes, at 230 cm/hour). The columnwas then equilibrated with a 50 mM sodium phosphate, pH 6.5 buffer (atleast 5 column volumes). Next the clarified ultrafiltered and dilutedmaterial (i.e. processed lysate APF fermentation material) was loaded at230 cm/hour onto the POROS® 50HQ anion exchange column, followed bywashing with at least about 20 column volumes of 50 mM sodium phosphate,pH 6.5 at 230 cm/hour until absorbance at 280 nm of column effluentdecreases to 0.10 AU, followed by eluting with 50 mM sodium acetate, pH4.8 at 230 cm/hour. The product pool was collected, when the absorbanceat 280 nm (A₂₈₀) increases to at least about 0.15 AU and through thepeak maximum to equal or less than about 0.2 AU on the trailing edge,into a vessel containing 1 column volume of 50 mM sodium acetate, pH4.8. This elution pool was stored at about 2° C. to about 8° C. for upto 48 hours.

The second chromatography step in the downstream process of this Example2 used a POROS® 20HS cation exchange chromatography resin packed into acolumn with an inner diameter of 8 cm and a column height of 5 cm. Theentire POROS® 20HS column operation was completed at ambienttemperature, and the flow was in the downward direction. The botulinumneurotoxin type A complex associates with the POROS® 20HS column resin.The botulinum neurotoxin type A complex was then eluted from the columnusing a salt step change. The product-related impurities were elutedwith the wash buffer and decontamination solution.

Particulars of the cation exchange step were: use of the POROS® 20HScolumn using 0.1 N sodium hydroxide solution for a minimum contact timeof 30 minutes (at least about 3 column volumes, at 230 cm/hour). Thecolumn was then equilibrated with a 50 mM sodium acetate, pH 4.8 buffer(at least about 5 column volumes). Next the POROS® 50HQ product pool(collected as described above, fresh or from refrigeration) was loadedonto the POROS® 20HS column. The column was then washed with a 50 mMsodium acetate, pH 4.8 buffer (at least about 3 column volumes) and thenwashed again with a 50 mM sodium acetate, 150 mM sodium chloride, pH 4.8buffer. The botulinum neurotoxin type A complex was eluted from thePOROS® 20HS column with a 50 mM sodium acetate, 250 mM sodium chloride,pH 4.8 buffer at 200 mL/min, the eluate was diverted into a bioprocesscollection bag (containing 1 column volume of 50 mM NaH₃C₂O₂, pH 4.8)when the A₂₈₀ increases to about 0.1 AU through peak maximum until theA₂₈₀ of the trailing edge of the elution peak decreases to a trailingedge value of ≦0.1 AU. The POROS® 20HS product pool was stored in thecollection bag at ambient temperature for up to about 6 hours.

In the three-column chromatography media process of this Example 2,eluent from the second (cation exchange) column was passed through a HICcolumn. The HIC column used was a Phenyl Sepharose HP hydrophobicinteraction chromatography resin packed into a column with an innerdiameter of about 8 cm and a column height of about 5 cm. The entirePhenyl Sepharose HP column operation was completed at ambienttemperature, and the flow was in the downward direction. The botulinumneurotoxin type A complex was eluted from the column using a decreasingsalt step change. The impurities were eluted during the load and withthe wash buffer and decontamination solution.

Particulars of the hydrophobic interaction chromatography step were: aPhenyl Sepharose HP column was initially sanitized with a 0.1 N sodiumhydroxide solution for a minimum contact time of 30 minutes (with atleast about 3 column volumes of a 0.1 N sodium hydroxide solution at 200cm/hour). The column was then equilibrated with at least about 5 columnvolumes of 50 mM sodium acetate, 0.4 M ammonium sulfate, pH 4.8 buffer.Next the POROS® 20HS (cation exchange column) product pool (from above)was combined 1:1 with a 50 mM sodium acetate, 0.8 M ammonium sulfate, pH4.8 buffer and loaded onto the Phenyl Sepharose HP column. The columnwas first washed with at least about 3 column volumes of a 50 mM sodiumacetate, 0.4 M ammonium sulfate, pH 4.8 buffer, and then washed with a50 mM sodium phosphate, 0.4 M ammonium sulfate, pH 6.5 buffer. Botulinumneurotoxin type A complex was eluted from the column with a 10 mM sodiumphosphate, 0.14 M ammonium sulfate, pH 6.5 buffer. The eluate wasdiverted into a bioprocess collection bag when the A₂₈₀ increased to0.05 AU. The eluate was collected until the A₂₈₀ of the trailing edge ofthe elution peak decreased to a value of 0.05 AU. The Phenyl SepharoseHP product pool was stored in the collection bag at ambient temperaturefor up to 6 hours.

A tangential flow filtration system was used to concentrate anddiafilter the Phenyl Sepharose HP chromatography step product pool intothe drug substance formulation buffer. Pall® Filtron Minimate cassetteswith a 100 kDa molecular weight cut off membrane were used for theconcentration and diafiltration steps. The formulated material was thenpassed through a Pall Mini Kleenpak® 0.2 μm filter to reduce thepotential bioburden. As stated previously, the UF/DF step concentratedthe Phenyl Sepharose HP product pool (eluent of the HIC column) to aBoNT/A complex concentration of 0.7 g/L and diafilters the concentratedmaterial with a 10 mM potassium citrate, pH 6.5 buffer.

Particulars of the ultrafiltration/diafiltration process used were asfollows. The UF/DF unit and Pall 100 kDa polyether sulfone membrane wasinitially flushed with a minimum of 5 L of water for injection (WFI) toremove the packing solution and sanitized with a minimum of 200 mL of a1 N sodium hydroxide solution under recirculation conditions for aminimum of 10 minutes, preferably at least 30 minutes, to sanitize theUF/DF unit. Next the membrane and UF/DF system were equilibrated withsufficient volumes of the 10 mM potassium citrate, pH 6.5 formulationbuffer until permeate and retentate pH was pH 6.5. After that the PhenylSepharose HP product pool was loaded onto the Minimate® tangential flowfiltration cassette and the HIC eluate concentrated to 0.7 g/L.Following the concentration step, the retentate pool was diafilteredagainst a minimum of 5 diafiltration volumes of the drug substanceformulation buffer (10 mM potassium citrate, pH 6.5) at a transmembranepressure of 7.5 psig (pounds per square inch gauge). The permeate outletwas then closed and the UF/DF system run for at least 2 minutes and thesystem rinsed with 50 mL of 10 mM potassium citrate, pH 6.5 formulationbuffer. After the rinse, the concentration of BoNT/A complex in theretentate pool was determined by measuring the offline A₂₇₈ and based onthe A₂₇₈ reading, the concentration of the retentate pool was adjustedto 0.5 g/L with 10 mM potassium citrate, pH 6.5 buffer. Theconcentration-adjusted retentate pool was then filtered through a PallMini Kleenpak 0.2 μm filter to reduce potential bioburden. The filteredconcentration-adjusted retentate pool was stored in a collection bag at2° C.-8° C. for up to 2 days.

The final purified botulinum neurotoxin type A complex obtained wasfilled into 1 mL Nunc® cryovials at 700 μL per vial and stored frozen.The filling operation was carried out in a class 100 biosafety cabinetat ambient temperature.

The downstream process (including use of 2 or 3 chromatography columns)was completed in only 1 to 3 days and the botulinum neurotoxin type Acomplex obtained was stored frozen in a potassium citrate, pH 6.5 bufferat a concentration of 0.5 g/L as a solution. In comparison, the priorart Schantz downstream (toxin purification) process uses multiplefiltration, precipitation, extraction and centrifugation steps to purifythe botulinum neurotoxin type A complex and requires 1-2 weeks tocomplete just the downstream steps, and the resultant drug substance(recovered botulinum neurotoxin) is stored refrigerated as an ammoniumsulfate suspension at a concentration of approximately 2.7 g/L. The useof chromatography instead of precipitation and the reduced processingtime resulted in a significantly improved, consistent downstreamprocess, as herein disclosed.

In accordance with one aspect, concentrations of vegetable-basedproducts, such as soy-based products, can be Soy Peptone Type II Hy-Soy®or SE50MK (a Kosher soy peptone) in culture and fermentation media.Hy-Soy® in the seed culture medium can range between 10-200 g/L.Preferably, the concentration of Hy-Soy® in the seed medium rangesbetween 15-150 g/L. Most preferably, the concentration of Hy-Soy® in theseed medium is approximately between about 20-30 g/L or an amounttherebetween. The concentration of glucose in seed medium can rangebetween 0.1 g/L and 20 g/L. Preferably, the concentration of glucoseranges between 0.5-15 g/L. Most preferably, the concentration of glucosein the culture medium is approximately 10 g/L. Yeast extract amounts canbe from about 5-20 g/L, more preferably from about 10-15 g/L or anamount therebetween. For example, the pH of the culture medium prior togrowth of Clostridium botulinum can be approximately pH 7.0-7.5, ortherebetween, preferably pH 7.3.

As an example, Hy-Soy® amounts in the production fermentation medium canrange between 10-200 g/L. Preferably, the concentration of Hy-Soy® inthe fermentation medium ranges between 15-150 g/L. Most preferably, theconcentration of Hy-Soy® in the fermentation medium is approximatelybetween about 20-40 g/L or an amount therebetween. The concentration ofglucose in fermentation medium can range between 0.1 g/L and 20 g/L.Preferably, the concentration of glucose ranges between 0.5-15 g/L or anamount therebetween. Not necessarily, but as above, the glucose can besterilized by autoclaving together with the other components of thefermentation medium. The pH level of the fermentation medium prior togrowth can be pH 7.0-7.8, preferably about 7.0-7.5 or therebetween, morepreferably pH 7.3.

As shown by the right hand side of FIG. 1, the two column APF processused in this Example 2 for obtaining a biologically active botulinumneurotoxin complex comprised the following steps: (a) culturingbacteria, such as Clostridium botulinum bacteria from an APF WCB vial,in a seed/culturing bottle, (b) then fermenting Clostridium botulinumbacteria in a fermentor (toxin production fermentor) having APFfermentation medium to expand the cell line, proceeding withfermentation and botulinum toxin production until a desired cell lysisphase is reached. Next, (c) harvesting (e.g. clarifying by filtration,)the APF fermentation medium to obtain a harvested fermentation medium,(d) proceeding with concentration and dilution resulting in a dilutedharvested fermentation medium that is (e) passed through a capturecolumn to remove impurities, (f) contacting eluent from the capturecolumn with a polishing column to further remove impurities, andoptionally a second polishing column (g) concentration and bufferexchange of the polishing column eluent, (h) followed by bioburdenreduction filtration and the (i) filling of vials.

In one example, the fermentation volume is 20 L, the total process timefor all steps was only 4 to 6 days, and high botulinum neurotoxin yieldwas obtained.

The following provides more details of a particular embodiment withinthe scope of our invention. The fermentation step was carried out in APFmedium using a 30 L stainless steel fermentor.

In this example below, a much-reduced volume of fermentation medium wasused while still providing a high yield of high potency botulinumneurotoxin type A complex. By using the following protocol, only 20 L orless, for example, of APF fermentation medium was required, instead ofthe typically larger, previous volumes (e.g. 115 L) of fermentationmedium required for producing commercially useful amounts for obtaininga botulinum neurotoxin.

The MACS anaerobic workstation (Don Whitley) with airlock provided anoxygen-deficient environment in which to manipulate anaerobic organisms.Access to and egress from the chamber was via a porthole system,comprised of inner and outer doors. The unit was temperature controlledto maintain a user setting within the chamber. A humidistat-controlledcondensing plate ensured the effective removal of excess moisture in thechamber. The chamber was illuminated for operator use and alarm for: lowgas pressure, continuous gas flow, and loss of power conditions. Thechamber was equipped with a HEPA filter to reduce viable and non viableparticulate levels in the anaerobic chamber. Anaerobic conditions weremaintained utilizing the “Anotox” and Palladium Deoxo “D” Catalystatmospheric scrubbing system. Condensate water from the condensing platewas collected and piped to an external reservoir where it is removed.

As disclosed above, an APF process was used for preparation of an APFWCB, having cell bank vials stored below −135° C. An APF WCB cell bankvial was thawed at room temperature for about 15 min before culturemedium inoculation, followed by a single cultivation step as disclosedabove to establish a “seed” culture. This was carried out in a modularatmospheric controlled system utilizing aseptic techniques throughout,to minimize bioburden. The modular atmospheric controlled system wascleaned before undertaking inoculation of the completed seed culturevial with APF WCB vial contents. Culture medium was prepared using 1 Nhydrochloric acid and 1 N sodium hydroxide (for pH adjustment), D(+)Glucose, Anhydrous (Mallinckrodt Baker, Cat#7730, 4.00 g), Soy PeptoneType II (SPTII) (Marcor, Cat #1130, 8.00 g), Water for Injection (WFI)400.0 mL and Yeast Extract (YE) (BD Cat #212730, 4.00 g). The soypeptone Type II and yeast extract solution was made by measuring 300 mLof WFI with a 500 mL graduated cylinder and poured into a seed culturebottle. The seed culture bottle was placed onto a stirrer and thestirrer activated. 8.00 g of SPTII and 4.00 g of yeast extract was addedto the seed culture bottle and mixed until dissolved. If dissolution wasnot complete after mixing, the mixture would be heated on low setting.The pH was measured and adjusted to about 7.30±0.05. The medium solutionwas brought up to about 360 mL with WFI. The seed culture bottle wasadequately vented to allow steam and gas transfer. A 10% Glucosesolution (w/v) was prepared by measuring about 30 mL of WFI with a 100mL graduated cylinder and placed into the pre-assembled glucose additionbottle, which was placed onto a stirrer and the stirrer activated. About4.00 g of glucose was added to the glucose addition bottle and mixeduntil dissolved (low heat was used if necessary to a dissolution) and qs(quantity sufficient) glucose solution to 40 mL with WFI. The glucoseaddition was then capped loosely with vent cap. Both the glucose andseed culture bottles are autoclaved at 123° C. for 30 minutes forsterilization. After sterilization, both items were removed from theautoclave and left to cool in a bio-safety cabinet. After coolingaseptically, 10% of the glucose solution was transferred into the seedculture bottle containing the yeast extract and soy peptone II solutionand mixed, thereby providing a completed seed culture bottle.

This completed seed culture bottle was placed into the pre-cleaned MACS(wherein a prepared anaerobic indicator was placed). The cap of thecompleted seed culture bottle was loosened. The completed seed culturebottle was then placed on a stir plate within the MACS (stir plateactivated to about 150 rpm) and the medium in the completed seed culturebottle was reduced for a minimum of 12 hours at about 34.5° C.+/−1° C.within the MACS, after which a 1 mL medium blank was sampled for opticaldensity measurement (for biomass determination at 540 nm). Afterwards,the completed seed culture bottle, in the MACS (anaerobic) wasinoculated. An APF WCB culture vial was obtained from the frozen cellbank and brought into the MACS. The vial was thawed for about 10-15minutes, after which about 400 μL of the vial contents were placeddirectly into the medium in the completed seed culture bottle. The capon the completed seed culture bottle was loosened completely and the capwas rested on top of the bottle and the stir pace was set to 150 rpm.After at least about 11 hours of incubation in the MACS, fermentationproduction was undertaken, as described below.

Probes (e.g. redox probe, pH probe, turbidity probe, e.g. by BroadleyJames and Optek) and sequence configuration of the fermentor, such as a30 L stainless steel fermentor, were checked and calibrated, andinserted into their respective fermentor ports and tightened in place.For example, a fermentor can be a ABEC 30 L (VT) Fermentor Systemconsisting of a 30 L volume fermentor vessel, an agitator drive system,piping assembly for utility connections (CIP, clean steam, CDA,Nitrogen, Oxygen, Process Chilled Water, bio-waste, and plant steam),instrumentation (pH, temperature, pressure, ReDox, optical density, andmass flow), and four peristaltic pumps. The bottom mounted agitatorspeed was controlled using an Allen-Bradley variable frequency drive(VFD). Semi-automatic and automatic control of the system is handled byan Allen-Bradley ControlLogix PLC with programming. The system wasdesigned to provide closed-loop PID (proportional-integral-derivative)control of culture temperature, pressure, pH, and redox duringfermentation operations. An Allen-Bradley DeviceNet® (an open devicelevel network) is utilized for control and communication with devicesand sensors on the skid.

For sterile hold, equilibrium, run and harvest modes, agitation,temperature, pressure and Nitrogen overlay are operated with thefollowing set points.

For sterile hold and equilibrium mode:

Controlled Parameter Set Points and Range Agitation 100 rpm ± 10Nitrogen Overlay 12 SLPM ± 2  Fermentor Pressure  5 psig ± 1 FermentorTemperature      35 ± 1° C. Redox −390 to −150 mVFor RUN mode:

Controlled Parameter Set Points and Range Agitation 70 rpm ± 5 NitrogenOverlay 12 SLPM ± 2  Fermentor Pressure  5 psig ± 1 FermentorTemperature      35 ± 1° C.For Harvest mode:

Controlled Parameter Set Points and Range Agitation 150 rpm ± 10Nitrogen Overlay 10 SLPM ± 2  Initial Fermentor Pressure 0 psigFermentor Temperature      25 ± 1° C.

To prepare fermentation medium, material needed include D(+) Glucose,Anhydrous (Mallinckrodt Baker, Cat#7730, 300.0 g), Soy Peptone Type II(SPTII) (Marcor, Cat #1130, 650.0 g), Water for Injection (WFI, 13 L)and Yeast Extract (YE) (BD Cat #212730, 240.0 g), along with standardbalances, a carboy (20 L, for example), glass bottle (5 L), graduatedcylinders, stir bars and stirrers. About 10 L of WFI were added into thecarboy along with a stir bar. The carboy was placed onto a stirrer andthe stirrer was activated, after which about 650.0 g of soy peptone typeII was added, along with about 240.00 g of YE. The fermentation mediumwas q.s. (quantity sufficient) to 13 L with WFI, and the carboy wascapped. A 10% glucose solution (w/v) was then prepared by adding about 2L if WFI into a glass 5 L bottle (with stir bar therein). Placed onto astirrer and with the bar spinning, about 300.00 g of glucose was addedinto the bottle, and mixed until dissolved. The glucose solution wasq.s. to 3 L with WFI and the bottled capped, thus providing a 10%glucose solution.

The fermentation medium in the carboy was added to the fermentor andpre-steam in place fermentor volume recorded and the fermentationsequence of operation was advanced. At the end of the SIP (steam inplace (122° C., +/−1° C.), the post-SIP fermentor volume was noted. Aglucose addition assembly, comprising a vessel having tube therefromwith and in-line 0.2 μm filter (PALL Corp.) and peristaltic pump, wasconnected to the fermentor and the line was subjected to SIP and allowedto cool. An addition valve port was opened and about 3 L of glucose(filter sterilized) was added, and the appropriate amount of WFI (filtersterilized) to q.s. the total fermentor volume to 20 L was added to theglucose addition bottle and pumped into the fermentor through the sameglucose filter line. The addition valve port was closed. The productionfermentation medium had its pH adjusted thereafter, to about pH7.3+/−0.05, with sterile 1 N sodium hydroxide or 1 N hydrochloric acid,utilizing SIP of addition lines, as required. Afterwards, parameters forsterile hold were set and held for about 12 hours before inoculation.The medium's starting glucose concentration was measured using ametabolite analyzer and glucose concentration recorded.

As stated above, at the end of seed culture incubation (about 11±1hours), 1 mL of sample was taken for optical density (OD) measurement.OD was measured offline at 540 nm using a spectrophotometer and ifwithin the appropriate range the OD value was recorded and culture wasused for fermentation. The fermentor turbidity probe was accordinglyzeroed. The seed inoculum bottle, from the anaerobic chamber, wasbrought over to the fermentor and a seed inoculum transfer assembly (aseed vessel with APF culture medium therein, the vessel having a cultureinoculum transfer line with a sterile Kleenpak™ Connector assemblyavailable from PALL Corp. or Millipore replaced the a valve of thefermentor, and tubing to Pump 1 was fixed. The fermentor pressure waslowered to 2 psig and entire volume of the seed inoculum bottle waspumped into the fermentor. At the end of inoculation, the onlineAbsorbance Units (AU) from the fermentor was recorded, fermentorparameters were set to RUN mode and time was recorded.

Fermentation then proceeded (fermentation runs can be from about 60hours to about 80 hours, preferably from about 68 hours to about 76hours, most preferably for about 72 hours) while samples were taken fromthe fermentor, at 24 and 48 hours, for example, while maintainingaseptic conditions. Tests that were run on at least one sample takenduring fermentation can include, but are not limited to, off-lineoptical density measurements, glucose measurements, ELISA, SDS-PAGE,Western blot, for example. At the end of the fermentation (end offermentation broth volume is from about 18-19 L, for example), a samplemay be taken (for testing by, for example, off-line optical densitymeasurements, glucose measurements, ELISA, SDS-PAGE, western blot andDNA/RNA quantification.

At the end of the fermentation, online optical density, EFT (elapsedfermentation time), and fermentation end time was recorded, as well asagitation rpm, temperature in ° C., pressure psig and Nitrogen overlayslpm and redox mV. Next, the production fermentation broth was subjectedto harvesting, i.e. the production fermentation broth is clarifiedthrough filtration whereby, for example, about 15 L of filtrate iscollected. The fermentation parameters were set for HARVEST and thefilter assembly for clarification was prepared (CUNO, 3M filtration)which includes a pre-filter, depth filter and at least one pressuregauge. The pre-filter and depth filter were flushed with about 20 L ofwater for injection. After flushing, the filtration assembly wasattached to the harvest/drain port of the fermentor. The fermentortemperature was decreased to about 25° C., after which clarification ofthe fermentation broth begins (record clarification start time, initialonline OD, initial pH, initial temperature and initial volume offermentor). The pressure in the fermentor was increased at a rate ofabout 1 psi (pound per square inch) about every 10 minutes duringfiltration, until a pressure of about 6 psi was reached, at which thepressure was held until the end of harvesting. This filter removesapproximately 80% of the RNA/DNA in the APF fermentation medium (theremainder essentially removed during later chromatography steps, asdiscussed below), thus doing away with prior reliance/use of RNaseand/or DNase to remove such components from the fermentation broth.Process parameters, such as pre-filter inlet pressure, depth filterinlet pressure, fermentor pressure, agitation and filtrate volume weremonitored at every 2 L of filtrate collected, at the end of which theclarification end time and volume of filtrate collected was recorded.Following completion of harvest step, the systems were decontaminatedand cleaned.

The filtrate carboy was brought into the BSC for sampling, from whichabout 0 mL of filtrate was sampled for offline OD measurements and otheranalysis (e.g. ELISA, SDS-PAGE, DNA/RNA and western blot).

The filtrate was then subjected to ultrafiltration/dilution. Atangential flow filter (TFF) unit assembly was assembled. The TFF unitwas rinsed for about 90 minutes with WFI at a preferred rate of about 2L per minute and then the TFF unit was sanitized by running 0.1 N sodiumhydroxide (re-circulated) therethrough for about 60 minutes, after which1 L of 10 mM sodium phosphate buffer, pH 6.5 was run therethrough,followed by a rinse with WFI for about 30 minutes. The filtrate from theharvest step (about 15 L) was then passed through the TFF (this iscarried out in a bio-safety cabinet), concentrating the filtrate down toabout 5 L+/−0.5 L (the concentration step proceeds at about 2 L perminute and at a trans-membrane pressure of about 5 psig). A sample ofthe permeate can be taken and subjected to ELISA, dsDNA, SDS-PAGE andwestern blot tests, for example. Once concentrated to about 5 L+/−0.5 L,the retentate pool was then diluted up to about 20 L with about 15 L ofsterile filtered 10 mM sodium phosphate buffer, pH 6.5, through the TFF,at about a rate of 2 L per minute. A sample can be then again be takenand subjected to ELISA, DNA/RNA, SDS-PAGE and western blot tests, forexample. The ultrafiltration/dilution material (retentate) was stored at4° C.

Following use all systems were decontaminated using either 1N sodiumhydroxide or sterilization (steam) temperatures and cleaned.

The following materials, equipment and procedures were used to make thesolutions, buffers, etc, set forth below for use in an exemplaryprocess, that is in the purification of the fermentation medium obtainedfrom the Example 2 processes so as to obtain a purified botulinumneurotoxin type A complex. Exemplary buffers utilized (filtered througha 0.2-micron vacuum filter and their conductivity measured in mS/cm, forrecordkeeping) include:10 mM sodium phosphate, pH 6.5; 50 mM sodiumphosphate, pH 6.5; 50 mM sodium acetate, pH 4.8; 50 mM sodium acetate,170 mM sodium chloride, pH 4.8; 50 mM sodium acetate, 250 mM sodiumchloride, pH 4.8; 50 mM sodium acetate, 1 M sodium chloride, pH 4.8; 50mM sodium acetate, pH 4.0 and 10 mM citrate, pH 6.5.

The following is an example of operations for purification and obtainingbotulinum neurotoxin type A from the Example 2 processes. Allproduct-contact parts were designed and constructed to ensure that theyare non-reactive and non-absorptive. Additionally, all equipment wasdesigned to allow the utilization of single use disposable systems orwas designed and constructed to facilitate sanitization, cleaning anddecontamination as per documented, validated methods. The systems orskids were designed to be non-product contacting while the flow pathsare designed to be single use disposable, including the chromatographycolumns and the all associated tubing. Chromatography components wereobtained from AlphaBio and UF/DF components were obtained from ScilogInc. The chromatography set ups used included a peristaltic pump forsolution delivery with variable speed drive, inlet valve manifold with 5inlets, a column valve manifold with an array of 3 automated valves,outlet valve manifold with 3 outlets, column effluent monitoring,including pH, conductivity, and UV, peak collection based on UVabsorbance, and instrumentation and controls required to complete thepurification operations. The control system had both the software andhardware designed to control the purification process. Commands and datawere entered via a HMI (Human Machine Interface) terminal. The operatorinitiated all automated process functions by commands at the HMI andmonitored and adjusted process parameters such as feed flow rates,pressure, conductivity, pH, UV absorbance and individual valvepositions.

The UF/DF system included of a recirculation pump, diafiltration pump, 2balances and a tangential flow filter (TFF) holder. The recirculationpump interfaced with 3 disposable pressure sensors and one of thebalances (located under the permeate reservoir) to control the flow rateto maintain a defined transmembrane pressure and stop, based on theweight of the permeate reservoir. The diafiltration pump interfaced withthe second balance (located under the retentate reservoir) to start andstop, based on maintaining a constant weight of the retentate reservoir.

After concentration and dilution of retentate material from theharvesting step (harvesting the animal protein free fermentationmedium), the material was loaded onto an anion exchange column. Thefollowing is the procedure used for packing and testing the anionexchange column useful in the Example 2 two column process.

Pre-packed columns were used for all three chromatographic steps. First,feed material (harvested APF media that had been subjected toultrafiltration/dilution) was passed through the anion exchange column(Poros 50HQ, from ABI as described above). At least 5 column volumes(CVs) of 50 mM sodium phosphate, pH 6.5, were utilized to equilibratethe anion exchange column (in this example, a capture column).

After equilibration, the loading step was performed, where feed material(post harvesting step harvested fermentation broth, of about 20 L, forexample)) was loaded onto the anion exchange column at a rate of about200 cm/hr for example. After 0.5 column volume of loaded material hadpassed through the anion exchange column, the flow through (FT) pool wascollected into a receptacle such as a polyethersulfone vessel, whiletoxin complex is bound to the anion exchange column material. This wasfollowed by a wash step, where at least about 15 column volumes of thewash buffer (e.g. 50 mM sodium phosphate at a pH of 6.5) was passedthrough the anion exchange column. The wash step was stopped when theUV, measured at the column outlet, in real time, decreased to less thanor equal to about 80 mAU. The wash buffer volume and the flowthrough/wash pool volume were recorded, and a 1 mL sample of the flowthrough/wash pool is taken and tested, for example, for toxinconcentration, nucleic acid content, whole cell proteins, SDS-PAGE,qPCR, 2D LC and ELISA.

The next step was the elution step, where elution buffer (e.g. 50 mMsodium acetate, pH 4.8) was pumped onto the anion exchange column. Whenthe UV reading at the column outlet, in real-time, increased to about150 mAU or more, collection of eluate in a container pre-filled with 1CV of elution buffer (50 mM sodium acetate, pH 4.8) was begun.Collection of eluate pool was stopped when the UV reading decreases toless than or equal to about 200 mAU (volume collected at this point isbetween about 1 to about 2 CVs). The chromatography system was thendecontaminated and cleaned using 1 N sodium hydroxide.

The eluate pool from the anion exchange column was then prepared foraddition onto the cation exchange column. The anion exchange eluatevolume, pH, conductivity and feed temperature were recoded and theeluate pool from the anion exchange column was diluted with 1 CV of 50mM sodium acetate, pH 4.8.

Following the run-through of the anion exchange column, cation exchangechromatography operation was undertaken. The cation exchange column(e.g. Poros® 20HS) was equilibrated with a minimum of 5 CVs ofequilibration buffer (50 mM sodium acetate, pH 4.8). Afterequilibration, the diluted eluate pool from the anion exchange columnwas loaded onto the cation exchange column and the total volume loadedwas recorded. After 0.5 column volume of loaded diluted eluate pool hadpassed through the cation exchange column, the flow through (FT) poolwas collected. A first wash of the cation exchange column was conductedwhere about 3-5 CVs of 50 mM sodium acetate, pH 4.8, was passed throughthe cation exchange column (volume of first wash buffer utilized wasrecorded). A second wash was performed, where about 3 CVs of 170 mMsodium chloride, 50 mM sodium acetate, pH 4.8, was pumped through thecolumn, this eluate being collected in a new container labeled “WASH 2Peak”. Collection was begun when the UV readings increase to greaterthan or equal to 50 mAU. 1 CV was collected and the second wash buffervolume utilized was recorded.

Elution of bulk toxin complex from the cation exchange column wascarried out utilizing elution buffer (e.g. 250 mM sodium chloride in 50mM sodium acetate, pH 4.8) which was pumped onto the cation exchangecolumn. When the UV reading of the elution reached at least about 100mAU, eluate collection begun into containers pre-filled with dilutionbuffers (40 mL of 100 mM potassium phosphate, pH 6.8 and 60 mL of 10 mMpotassium citrate, pH 6.5). Collection of eluate from the cationexchange column continued until UV readings decreased to about 100 mAUor less. The total volume of elute, after dilution, was recorded. Thecation exchange chromatography system was then decontaminated andcleaned.

Following elution from the cation exchange column, the eluate wassubjected to filtration. A tangential flow filtration (TFF) system wasutilized, using three 100K MWCO membranes (Sartorius A G, Goettingen,Germany) stacked one atop the other. The cation exchange eluate poolinitial volume was noted, as are the diafiltration/equilibration andsanitation solution descriptions. For example, the diafiltrationsolution can be 10 mM potassium citrate, pH 6.5 and the sanitationsolution can be 0.1 N sodium hydroxide. System set up proceeded withconnection of one tube from the reservoir containing either eluate fromthe cation column (IAPF) or HIC column (FAPF), the eluate containingbotulinum toxin, through the ultrafiltration pump head into the inlet ofthe tangential flow filtration membrane. A second tube from the permeateoutlet of the tangential flow filtration membrane was connected to theultrafiltration (UF) permeate container. A tube from the retentateoutlet of the tangential flow filtration membrane to the retentatereservoir was secured, and a fourth tube from the diafiltration (DF)buffer through the diafiltration pump head and into the retentatereservoir was also secured. The storage buffer of the system wasflushed, as is the membrane, by flushing the membrane with at leastabout 720 mL of water for injection (WFI) with the retentate directed towaste, after which the membrane was further flushed with at least about4200 mL of water for injection with the retentate recirculating to thereservoir. After this, membrane sanitation (if necessary) was carriedout by flushing the membrane with at least about 200 mL of 1N sodiumhydroxide with the retentate directed to waste, followed by a flushingof the membrane with at least about 200 mL of 1N NaOH with the retentaterecirculating to the reservoir for a minimum of 30 minutes.Equilibration was then performed, by flushing the membrane withequilibration buffer (10 mM potassium citrate at a pH of 6.5), withretentate directed to waste until the retentate and permeate pH waswithin +/−0.2 units of the pH of the equilibration buffer (for example,within +/−0.2 units of pH 6.5).

The concentration of the material (eluate (product pool) from the cationexchange column) was determined, to see if dilution or concentration(exemplary processing) was appropriate (an example target concentrationcan be about 0.7 mg/mL). Dilution was accomplished utilizing 10 mMpotassium citrate, pH 6.5. A target volume was determined, for examplefor a 0.7 mg/mL product concentration (target vol=(startingconcentration/starting vol)/0.7 mg/mL).

The product pool (eluate (accordingly processed or not) from cationexchange column) was loaded onto the membrane and recirculation (withpermeate outlet closed) of the system (TFF system) was run for at least2 minutes with no backpressure, after which the permeate valve wasslowly opened while adjusting the retentate back pressure valve to atarget of about 7 psig transmembrane pressure. For dilution, 10 mMpotassium citrate, pH 6.5 is added to target volume, and moved ontodiafiltration without ultrafiltration; for concentration,ultrafiltration is begun. For diafiltration: permeate waste wascollected in a new container (target diafiltration volume is 5×diafiltration volume) and diafiltered with at least 5 diafiltrationvolumes of 10 mM potassium citrate, pH 6.5. Diafiltration process datawas collected at a minimum of 10-minute intervals (permeate weight g/volmL, inlet pressure (psig), retentate pressure (psig), permeate pressure(psig) and transmembrane pressure (psig)). For recirculation/and rinse:with the permeate outlet filter closed, the system was recirculated/runfor at least 2 minutes with no backpressure and the system was rinsedwith at least 20 mL of 10 mM potassium citrate, pH 6.5. The product poolincludes the retentate and the rinse. A sample can be taken from theproduct pool and subjected to verification analysis including, forexample, UV at 278 nm, SDS-Page, LcHPLC. SE-HPLC, qPCR, RP-HPLC,Native-Page, AUC, Limulus amebocyte lysate, Western Blot and ELISAtests. For post-use cleaning, the system was flushed with 1N sodiumhydroxide, recirculated for at least 10 minutes, after which the systemwas flushed and stored with 0.1 N sodium hydroxide therein.

Sterile filtration and filling was then conducted for storing anddividing the bulk neurotoxin. Concentration adjustment was performed toadjust toxin concentration, using 10 mM potassium citrate, pH 6.5, toabout 0.5 mg/mL with the post rinse sample. If toxin concentration wasless than about 0.5 mg/mL, then no concentration adjustment is needed.

Using a sterile pipette, 10 mL/0.75 mL aliquots into each of sterile 15mL/1.5 mL sample tubes were made. The product container was gentlystirred by hand and transfer the required amount of solution (containingbulk drug substance, i.e. bulk botulinum toxin) into each vial. Thesamples were stored a maximum of 5 days at 2° C.-8° C. refrigerator or0.75 mL of the filtrate product pool was transferred to cryovials. Thecryovials are stored at −70° C.+/−5° C.

Example 3 Compounding Method

A pharmaceutical composition suitable for administration to a patientcan be made by compounding a botulinum neurotoxin obtained from anExample 2 process with one or more excipients. An excipient can act tostabilize the botulinum toxin during the compounding process and duringa subsequent period of storage before use. An excipient can alsofunction as a bulking agent and/or to provide a certain tonicity to thepharmaceutical composition. Compounding requires a many fold dilution ofthe botulinum neurotoxin obtained from an Example 2 process, mixing withone or more excipients (such as albumin [such as a human serum albuminor a recombinant human albumin] and sodium chloride) to thereby form atoxin composition, and preparation of a storage and shipment stable formof the toxin composition, as by lyophilizing, freeze drying or vacuumdrying the composition. Thus, about 1.5 to 1.9 ng of the Example 2obtained botulinum toxin type A complex is compounded with about 0.5milligrams of recombinant human albumin (Delta Biotechnologies) andabout 0.9 milligram of sodium chloride by mixing these three ingredientstogether followed by vacuum drying. Vacuum drying can take place fromabout 20° C. to about 25° C., at a pressure of about 80 mm Hg, for about5 hours, at which time vials in which these components are vacuum driedare sealed under vacuum and capped, thereby obtaining a vial with about100 units of botulinum neurotoxin type A complex. The resulting solid(powdered) vacuum dried product is, upon use, reconstituted with normal(0.9%) saline and used to treat patients with various indications, suchas cervical dystonia and hyperhidrosis. Lyophilizing, vacuum or freezedrying prepares a storage and shipment stable form of the compoundedbotulinum neurotoxin.

In another example, from about 1.5-1.9 ng of the bulk botulinum toxintype A is compounded with about 0.5 milligrams of human serum albumin(Baxter/Immuno, Octapharma, and Pharmacia & Upjohn) and about 0.9milligram of sodium chloride by mixing these three ingredients togetherfollowed by vacuum drying. Exemplary vacuum drying can take place fromabout 20° C. to about 25° C., at a pressure of about 80 μm Hg, for about5 hours, at which time the vials in which these components are vacuumdried are sealed under vacuum and capped, thereby obtaining a vial withabout 100 units of botulinum toxin. The resulting solid (powdered)vacuum dried product is, upon use, reconstituted with normal (0.9%)saline and used to treat patients with various indications, such ascervical dystonia and hyperhydrosis. Additionally, a pharmaceuticalbotulinum toxin composition can contain human serum albumin and/orlactose for example. In one example, about 1.5-1.9 ng of the bulkbotulinum toxin type A can be compounded with about 125 micrograms ofhuman serum albumin, and 2.5 milligrams of lactose and vacuum dried,lyophilized or freeze dried for storage stability, for example. In stillanother example, about 1.5-1.9 ng of botulinum neurotoxin obtained bythe processes disclosed herein can be combined with about 10 mg oftrehalose and about 0.5 mg of serum albumin (such as human serumalbumin, native or recombinant), and optionally, about 1 milligram ofmethionine to provide about 100 units of botulinum toxin dried product.This composition can be lyophilized and be reconstituted later with,before use, about 1 mL of distilled sterile water or sterile unpreservedsaline (0.9% sodium chloride for injection), for example. In particularexamples, pharmaceutical botulinum toxin compositions can includesucrose, such as in an exemplary formulation having about 1.5-1.9 ng ofbotulinum neurotoxin obtained by the processes disclosed herein combinedwith human serum albumin 20% and sucrose, which can also be lyophilizedto provide about 100 units of botulinum toxin type A, and laterreconstituted with unpreserved saline (in a volume of about 0.5 mL toabout 8.0 mL for example). In a particular example, 200 units ofbotulinum neurotoxin can be combined with about 10 mg of sucrose and 2mg of human serum albumin per mL, and the resultant composition placedinto vials and freeze-dried, to be later reconstituted before use withphysiological saline.

Additionally, compounding can also utilize the neurotoxic component(i.e. the about 150 kDa component of the botulinum toxin type A complex,free of complexing proteins) of the botulinum toxin type A complexobtainable by the IAPF processes herein disclosed. In one method ofpurifying the about 150 kDa neurotoxic component from the associatednon-toxic proteins (e.g. HAs, NTNH), type A neurotoxin is purified fromthe associated non-toxic proteins of the complex by a modification ofthe method of Tse et al. (1982) (Goodnough, M. C., 1994, Thesis, UW,Wis.). Botulinum neurotoxin complex obtained by our IAPF process (whichutilizes either the 2-column anion-cation or 3-column anion-cation-HICsteps, as discussed above) is recovered from an DEAE-Sephadex A 50(Sigma Chemical Co., St. Louis, Mo.), pH 5.5, column and is precipitatedby addition of 39 g of solid ammonium sulfate/100 mL. The precipitatedtoxin complex is collected by centrifugation, dialyzed against 25 mMsodium phosphate, pH 7.9, and applied to a DEAE-Sephadex A50 columnequilibrated with the same buffer. The neurotoxic component is separatedfrom the non-toxic proteins of the complex and eluted from the columnwith a linear 0-0.5 M sodium chloride gradient. Partially purifiedneurotoxin component is recovered from the DEAE-Sephadex A50 column atpH 7.9 and dialyzed against 25 mM sodium phosphate, pH 7.0. The dialyzedtoxin is applied to SP-Sephadex C50 (Sigma Chemical Co.) in 25 mM sodiumphosphate, pH 7.0. Contaminating material does not bind to the columnunder these conditions. The pure neurotoxin (the about 150 kDacomponent) is eluted with a linear 0-0.25 M sodium chloride gradient.The about 150-kDa pure neurotoxin can be further purified by metalaffinity chromatography, gel filtration or other methods of proteinchromatography. As above, this pure neurotoxin (the about 150 kDaneurotoxic component of a botulinum toxin complex) can be lyophilized,vacuum or freeze-dried with the various excipients (e.g. serum albumin,sucrose, lactose, sodium chloride, trehalose, etc.) discussed above.

The bulk botulinum neurotoxin complex obtained by our IAPF process, canbe compounded in numerous ways. Exemplary patents that disclose variousformulations of botulinum toxins, such as U.S. Pat. No. 6,087,327(discloses a composition of botulinum toxin types A and B formulatedwith gelatin); U.S. Pat. No. 5,512,547 (Johnson et al) entitled“Pharmaceutical Composition of Botulinum Neurotoxin and Method ofPreparation” issued Apr. 30, 1996 and claims a pure botulinum type Aformulation comprising albumin and trehalose, storage stable at 37° C.;U.S. Pat. No. 5,756,468 (Johnson et al) issued May 26, 1998(“Pharmaceutical Compositions of Botulinum Toxin or Botulinum Neurotoxinand Method of Preparation”), and claims a lyophilized botulinum toxinformulation comprising a thioalkyl, albumin and trehalose which can bestored between 25° C. and 42° C.; U.S. Pat. No. 5,696,077 (Johnson etal) entitled “Pharmaceutical Composition Containing Botulinum B Complex”issued Dec. 9, 1997 and claims a freeze dried, sodium chloride-freebotulinum type B complex formation comprising a type B complex and aprotein excipient; and U.S. patent application publication number 20030118598 (Hunt) discloses uses of various excipients such as arecombinant albumin, collagen or a starch to stabilize a botulinum toxin(all of these published U.S. patent applications or U.S. patents arehereby incorporated by reference in their entirety), all provideexamples of various useful formulations/excipients that may be used tocompound the bulk botulinum neurotoxin provided by our IAPF process andprovide a pharmaceutical composition.

The botulinum toxin complex obtained can be eluted from an ion exchangecolumn in a pH 7-8 buffer to disassociate the non toxin complex proteinsfrom the botulinum toxin molecule, thereby providing (depending upon thetype of Clostridium botulinum bacterium fermented) botulinum toxin typeA neurotoxic component with an approximately 150 kDa molecular weight,and a specific potency of 1-2×10⁸ LD₅₀ U/mg or greater; or purifiedbotulinum toxin type B with an approximately 156 kDa molecular weightand a specific potency of 1-2×10⁸ LD₅₀ U/mg or greater, or purifiedbotulinum toxin type F with an approximately 155 kDa molecular weightand a specific potency of 1-2×10⁷ LD₅₀ U/mg or greater.

Our invention provides many benefits. Firstly, the two and three columnprocesses of Example 2 eliminates the use of animal source reagents andmedia (e.g. casein hydrolysate and Columbia blood agar plates) thusmarkedly decreasing the theoretical risks of patient exposure toprion-like agents or other infectious agents. Secondly, the two andthree column chromatographic processes (and associated systems andapparatus) of example 2 are highly reproducible, as evidenced byexcellent batch to batch consistency. This improvement translates to amore consistent clinical profile in patients who require repeatedtreatments with commercially available botulinum toxin containingcompounds over several years. Analytical studies of drug substance(botulinum neurotoxin) from the herein disclosed IAPF processes (2 and 3column) revealed a lower load of protein and nucleic acid impurities.This lower load of protein impurities translates into a lower risk ofimmunogenicity (antibody production). In addition, the improved purityof the IPAF process translates into a lower incidence of thenon-specific symptoms commonly associated with biologic drugs (eg,nasopharyngitis, upper respiratory tract symptoms, musculoskeletalsymptoms, headache, etc.). Furthermore, the improved downsized scale ofthis process decreases the risk of BoNT/A exposure in laboratory andmanufacturing facility staff.

Exemplary advantages of the present invention include, for example:

1. Safety is improved since no component or substance derived fromanimal source (e.g. human or animal) is used in the process, use ofDNase and RNase, Columbia blood agar plates, casein is eliminated(replaced, for example, by: charged filtration during theclarification/harvesting step and modern chromatography techniques; byseeding culture media directly with cells from a working cell bank, thatis, cells previously selected and propagated/maintained in APF media;and culture bottle and fermentation media replaced with Soy Peptone TypeII (SPTII) as a peptone source).

2. Between about 50 mg to about 200 mg of high quality botulinum toxintype A complex can be obtained per 10 L of fermentation medium.

3. The purified bulk toxin is obtained from a process which is robust,consistent, scalable, validatable, and cGMP compliant. Robust means theprocess is reproducible even upon an about ±10% change in one or more ofthe process parameters. Validatable means the process reproduciblyprovides consistent yields of purified toxin. cGMP compliance means thatthe process can be easily converted to a manufacturing process thatcomplies with FDA required current Good Manufacturing Practices.

4. The potency of the final purified botulinum toxin complex meets orexceeds the potency (e.g. as determined by the MLD50 assay) of purifiedbotulinum toxin complex obtained from a Schantz or modified Schantzprocess.

5. Replacement of any precipitation steps with chromatographic steps topurify a bulk botulinum toxin complex, which improves the specificity ofthe purification process.

6. New improved process facilitates reduction of scale resulting inimproved handling and achievement of an operational success rate of >95%(for example, reduced from typical volumes utilizing 110 L-120 L offermentation media down to about 10 L to about 50 L, even down to about2 L to about 30 L of fermentation media or an amount therebetween.Typical current production scale for bulk drug substance is 115 L ofnon-APF fermentation medium, and has, as one aspect of our invention,been reduced to 20 L of fermentation medium. This reduction in scale ismade possible by optimizing the synthesis and cellular release of theBoNT/A complex as well as overall yield across the purification steps,resulting in similar quantity of final bulk botulinum toxin (drugsubstance) as obtained in prior processes requiring, for example 5× oreven more fermentation volumes (e.g. 115 L). This reduced scalefacilitates easier management of the fermentation working volume andthus minimizes the potential risk of operator exposure to the BoNT/Acomplex, an important operational and safety advantage.

7. Due to the potentially lethal nature of the BoNT/A complex, closedsystems have been implemented throughout the manufacturing process asherein disclosed. Unlike prior art methods, no drug substance materialproduced in accordance with aspects of the present invention is exposedto the environment during transfer between unit operations; alloperations are wholly contained.

8. The bulk botulinum toxin manufacturing process herein disclosed issimplified at all steps without sacrificing the identity, quality,purity, or potency of the drug substance during manufacture. A number ofsteps utilized in a non-APF process have been eliminated in theredesigned IAPF process, thereby reducing production time from, forexample, 21 days to 6 days or less.

9. The storage condition of bulk botulinum toxin as a frozen solutiongreatly improves drug substance stability.

Various publications, patents and/or references have been cited herein,the contents of which, in their entireties, are incorporated herein byreference. Groupings of alternative elements or embodiments of theinvention disclosed herein are not to be construed as limitations. Eachgroup member may be referred to and claimed individually or in anycombination with other members of the group or other elements foundherein. It is anticipated that one or more members of a group may beincluded in, or deleted from, a group for reasons of convenience and/orpatentability. Moreover, any combination of the above-described elementsin all possible variations thereof is encompassed by the inventionunless otherwise indicated herein or otherwise clearly contradicted bycontext.

Although the present invention has been described in detail with regardto certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.Accordingly, the spirit and scope of the following claims should not belimited to particular descriptions of the embodiments set forth above.

1. A substantially animal product free (APF) process utilizingchromatography for obtaining a botulinum neurotoxin, the processcomprising the following steps: (a) providing a substantially APFfermentation medium; (b) fermenting a non-clostridial species in thefermentation medium, wherein the non-clostridial species produces abotulinum neurotoxin, and; (c) recovering the botulinum neurotoxin fromthe fermentation medium by contacting the fermentation medium with ananion exchange chromatography media followed by contacting an eluentfrom the anion exchange chromatography medium with a cation exchangechromatography medium, thereby obtaining the botulinum neurotoxin fromthe substantially APF chromatography process.
 2. The process of claim 1,wherein the botulinum neurotoxin obtained comprises one part or lessresidual nucleic acid per million of the botulinum neurotoxin obtained.3. The process of claim 1, wherein the process is carried out in oneweek or less.
 4. A botulinum neurotoxin obtained by the process ofclaim
 1. 5. A substantially animal product free (APF) process utilizingchromatography for obtaining a botulinum neurotoxin, the processcomprising the following steps: (a) providing a substantially APFfermentation medium; (b) fermenting a non-clostridial species in thefermentation medium, wherein the non-clostridial species produces abotulinum neurotoxin, and; (c) recovering the botulinum neurotoxin fromthe fermentation medium by contacting the fermentation medium with ananion exchange chromatography media followed by contacting an eluentfrom the anion exchange chromatography medium with a hydrophobicinteraction chromatography medium, thereby obtaining the botulinumneurotoxin from the substantially APF chromatography process.
 6. Theprocess of claim 5, wherein the botulinum neurotoxin obtained comprisesone part or less residual nucleic acid per million of the botulinumneurotoxin obtained.
 7. The process of claim 5, wherein the process iscarried out in one week or less.
 8. A botulinum neurotoxin obtained bythe process of claim 5.